Whi3 mnemon association with endoplasmic reticulum membranes confines the memory of deceptive courtship to the yeast mother cell

Prion-like proteins are involved in many aspects of cellular physiology, including cellular memory. In response to deceptive courtship, budding yeast escapes pheromone-induced cell-cycle arrest through the coalescence of the G1/S inhibitor Whi3 into a dominant, inactive super-assembly. Whi3 is a mnemon (Whi3(mnem)), a protein that conformational change maintains as a trait in the mother cell but is not inherited by the daughter cells. How the maintenance and asymmetric inheritance of Whi3(mnem) are achieved is unknown. Here, we report that Whi3(mnem) is closely associated with endoplasmic reticulum (ER) membranes and is retained in the mother cell by the lateral diffusion barriers present at the bud neck. Strikingly, barrier defects made Whi3(mnem) propagate in a mitotically stable, prion-like manner. The amyloid-forming glutamine-rich domain of Whi3 was required for both mnemon and prion-like behaviors. Thus, we propose that Whi3(mnem) is in a self-templating state, lending temporal maintenance of memory, whereas its association with the compartmentalized membranes of the ER prevents infectious propagation to the daughter cells. These results suggest that confined self-templating super-assembly is a powerful mechanism for the long-term encoding of information in a spatially defined manner. Yeast courtship may provide insights on how individual synapses become potentiated in neuronal memory.Peer reviewe

Functional and structural studies of bioactive peptides: From cell penetration to aggregation and structure stabilization

Peptides and proteins have many functions and are involved in almost every aspect of life. They catalyze reactions, act as receptors or signaling molecules, and serve as structural scaffolds in cells. Their functional roles are in most cases indissociable from the structure they adopt. In this thesis, three interesting features of peptides and proteins were investigated, namely the cell-penetrating capabilities of short peptides derived from the nitrogen storage polymer cyanophycin, the stabilizing effects of N-methyl asparagine (Asm) residues on pore forming structures, and the amyloidogenic properties of the yeast protein Whi3, which is believed to be involved in single cell memory. The cellular membrane defines the boundaries of the cell and regulates trafficking of molecules into and out of the cytoplasm. In fact, the cell membrane is one of the hardest barriers to cross in nature, which makes delivery of many therapeutic agents very challenging. For this reason, cell-penetrating peptides (CPPs) have attracted the interest of the scientific community, since they can be used as vectors to transport diverse cargos into cells. Although the exact mechanisms these peptides use to pass through membranes are not completely understood, CPPs typically contain multiple arginine (Arg) residues. The polymer cyanophycin, which is composed of Arg residues appended to a poly-aspartate backbone, is interesting in this context. In its polymeric form, it is too large to cross membranes, but we wondered whether shorter segments might. Because the cyanophycin building blocks contain both carboxylate and guanidinium groups in close proximity, they possess zwitterionic character unlike common CPPs. To study how these charges affect cell-penetrating properties, we synthesized three octameric cyanophycin derivatives. One variant retained the free carboxylates of the natural polymer, and the negative charges were removed in the other two by converting the carboxylates to methyl esters or amides. Interestingly, only the variants that had a net positive charge were found to penetrate into cells. Unfortunately, these derivatives were also quite toxic. In contrast, the zwitterionic cyanophycin analog exhibited neither cell-penetrating activity nor toxicity. The inactivity of this variant is likely due to neutralization of the positively charged guanidinium groups by formation of intramolecular salt bridges with the internal carboxylates. To test this hypothesis, the octamer was investigated by 1H-NMR and CD spectroscopy, but the peptide appears to be largely unstructured in solution. A net positive charge is evidently critical for internalization of standard CPP peptides but accompanying toxicity, most likely caused by perturbation of membranes, may well reduce the scope of potential therapeutic applications. CPPs are not the only compounds that interact with cellular membranes. Indeed, numerous peptides are known to insert into membranes and form defined pores, thereby disrupting the osmotic gradient generated by the bilayer. One of these pore-forming peptides, polytheonamide B, displays extensive post-translational modification. Nearly 50% of its residues are modified, some hydroxylated, others methylated, and many epimerized. Methylation of the Asn side chains that form a stripe along one face of the polytheonamide -helix is particularly striking. Molecular dynamics (MD) simulations suggested that this modification plays a crucial role in stabilizing the pore, while the other modifications have no impact. To provide experimental support for this hypothesis, we chemically synthesized several peptides with and without Asm residues and studied their properties. The first peptide we studied was the antibiotic gramicidin A (GramA), which forms -helices like polytheonamide. We replaced five residues with either Asm (GramA-Asm) or Asn (GramA-Asn). MD simulations indicated that the Asm residues would stabilize the GramA-Asm pore compared to wild-type GramA, whereas Asn would destabilize it. However, contrary to these predictions, NMR and CD spectroscopy showed that the synthetic Asm-containing peptide was largely unstructured both in solution and in micelles. In fact, the Asn variant seemed more structured than its Asm counterpart. The well-studied cyclic peptide cyclo[(L-Phe-D-MeN-D-Ala)4], which assembles as dimeric pore-like -sheet structures, provided a simpler system to investigate whether Asm residues can stabilize -sheets. We successfully synthesized several derivatives where one, two or four Phe residues were replaced by Asm or Asn residues (cyclo-Asm/Asn-1, cyclo-Asm/Asn-2 and cyclo-Asm/Asn-4, respectively), as well as one variant with a Cys residue for disulfide bridge formation (cyclo-Cys_Asm/Asn-1). Although only the cyclo-Asm/Asn-1 and cyclo-Asm/Asn-4 peptides have been biophysically characterized as yet, preliminary NMR results already suggest that cyclo-Asm-1 has stronger H-bonding interactions than its unmethylated counterpart cyclo-Asn-1. Preliminary data for cyclo-Asm/Asn-4, on the other hand, showed that both compounds are unexpectedly floppy. More work will be needed to complete the characterization of this system, but it seems that N-methylation of the Asn side chain can influence peptide structure in favorable cases. Whether such effects can be harnessed to stabilize specific conformations more generally is less clear, however. The final project in this thesis addressed the putative priogenic properties of the yeast protein Whi3. Amyloid fibers have long been associated with degenerative diseases, like Alzheimer’s or Huntington’s disease. However, amyloid formation can sometimes be useful, for example as a mean of regulating protein function. Whi3 forms asymmetrically segregated superassemblies that correlate with the escape of pheromone-induced cell-cycle arrest, and was predicted to be a prion based on two regions rich in Gln and Asn residues (polyQ and polyN). When these regions were deleted, aggregation was suppressed and cells could no longer escape pheromone-induced cell-cycle arrest. These observations led to the hypothesis that Whi3 is a “memory prion” (mnemon). To test this hypothesis, we investigated the aggregation behavior of full-length Whi3 in cells and characterized chemically synthesized peptides corresponding to its polyQ domain in vitro. For in vivo studies with full-length Whi3, cell lysate containing epitope-tagged Whi3, with and without the amyloidogenic polyQ region, was produced in the presence and absence of pheromone. Immunostaining analysis of Western blots from sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and semi-denaturating detergent agarose gels (SDD-AGE) showed that full-length Whi3 disappeared from samples treated with pheromone, consistent with enhanced aggregation. The fact that this phenotype was less pronounced with Whi3 variants lacking the polyQ region supports the notion that this segment is critical for the formation of the segregated superassemblies. Importantly, the aggregated protein could be rescued by boiling the samples in SDD-AGE, and could also be used to seed the aggregation of unaggregated Whi3 proteins. The polyQ segment itself was chemically synthesized by SPPS for independent confirmation of its amyloidogenic properties. It exhibited the typical characteristics of amyloids, forming fibrillar structures upon incubation in buffer solution. Fiber formation, which was monitored with the fluorescence dye bisANS, displayed standard sigmoidal growth curves and could be speeded up by seeding. The resulting fibrillar structures showed considerable polymorphism when analyzed by electron microscopy (EM). To study the effect of phosphorylation on Whi3 aggregation, elongated variants of the polyQ peptide containing selectively phosphorylated Ser residues were prepared by KAHA ligation of two synthetic fragments. In contrast to the unphosphorylated polyQ peptides, which formed large fibrillar assemblies with clearly distinguishable hierarchies, the phosphopeptides only gave unstructured aggregates. Although polyQ amyloids could be used as seeds to promote fibrillization of other polyQ peptides, they did not induce aggregation of the full-length Whi3 protein, suggesting that formation of the segregated superassemblies observed in vivo may depend on more than the polyQ region alone. The three projects described in this thesis highlight the utility of synthetic peptides for addressing a range of interesting biological questions. The use of SPPS enables virtually any modification of a peptide backbone and its side chains. As such, derivatives can be produced that would be otherwise difficult or impossible to obtain by recombinant technologies, providing powerful tools for dissecting the chemical origins of biological properties

A cryptic third active site in cyanophycin synthetase creates primers for polymerization

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.ISSN:2041-172

Structure and function of a hexameric cyanophycin synthetase 2

Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide β-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.ISSN:1469-896XISSN:0961-836

Chiral recognition in amyloid fiber growth

PMID: 26929241Insoluble amyloid fibers represent a pathological signature of many human diseases. To treat such diseases, inhibition of amyloid formation has been proposed as a possible therapeutic strategy. d-Peptides, which possess high proteolytic stability and lessened immunogenicity, are attractive candidates in this context. However, a molecular understanding of chiral recognition phenomena for d-peptides and l-amyloids is currently incomplete. Here we report experiments on amyloid growth of individual enantiomers and their mixtures for two distinct polypeptide systems of different length and structural organization: a 44-residue covalently-linked dimer derived from a peptide corresponding to the [20-41]-fragment of human β2-microglobulin (β2m) and the 99-residue full-length protein. For the dimeric [20-41]β2m construct, a combination of electron paramagnetic resonance of nitroxide-labeled constructs and (13) C-isotope edited FT-IR spectroscopy of (13) C-labeled preparations was used to show that racemic mixtures precipitate as intact homochiral fibers, i.e. undergo spontaneous Pasteur-like resolution into a mixture of left- and right-handed amyloids. In the case of full-length β2m, the presence of the mirror-image d-protein affords morphologically distinct amyloids that are composed largely of enantiopure domains. Removal of the l-component from hybrid amyloids by proteolytic digestion results in their rapid transformation into characteristic long straight d-β2m amyloids. Furthermore, the full-length d-enantiomer of β2m was found to be an efficient inhibitor of l-β2m amyloid growth. This observation highlights the potential of longer d-polypeptides for future development into inhibitors of amyloid propagation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd

Surface-Engineered Cationic Nanocrystals Stable in Biological Buffers and High Ionic Strength Solutions

Progress in colloidal synthesis in the last two decades has enabled high-quality semiconductor, plasmonic, and magnetic nanocrystals (NCs). As synthesized, these NCs are usually capped with long-chain apolar ligands. Postsynthetic surface functionalization is required for rendering such NCs colloidally stable in polar media such as water. However, unlike small anionic molecules and polymeric coatings, producing positively charged stable NCs, especially at high ionic strengths, has remained challenging. Here, we present a general approach to achieve aqueously stable cationic NCs using a set of small (<2.5 nm long) positively charged ligands. The applicability of this method is demonstrated for a variety of materials including semiconductor CdSe/CdS core/shell NCs, magnetic Fe@Fe3O4, Fe3O4, and FePt NCs, and three different classes of plasmonic Au NCs including large nanorods. The obtained cationic NCs typically have zeta potential values ranging from +30 to +60 mV and retain colloidal stability for days to months, depending on NC/ligand pair, in several biological buffers at elevated pH and in concentrated salt solutions. This allowed us to demonstrate site-specific staining of cellular structures using fluorescent cationic NCs with several different surface chemistries. Furthermore, colloidal stability of the obtained NCs in the presence of other charged species allowed the assembly of cationic and anionic counterparts driven primarily by electrostatic attraction. With this approach, we prepare highly uniform 3D and 2D binary mixtures of NCs through induced homogeneous aggregation and alternating-charge layer-by-layer deposition, respectively. Such binary mixtures may provide a new route in the engineering of nanocrystalline solids for electronics, thermoelectrics, and photovoltaics.ISSN:0897-475

Covalent Tethering and Residues with Bulky Hydrophobic Side Chains Enable Self-Assembly of Distinct Amyloid Structures

PMID: 27717158Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20-41] fragment of human β2-microglobulin into fibers with either trimeric or dimeric amyloid cores. Using a set of biophysical and biochemical methods we demonstrated their distinct structural, morphological, and templating properties. Furthermore, we showed that supramolecular approaches in which the peptide is modified with bulky substituents can also be applied to modulate the formation of different fiber polymorphs. Such strategies, when applied to disease-related peptides and proteins, will greatly help in the evaluation of the biological properties of structurally distinct amyloids

Cell Penetration, Herbicidal Activity, and in‐vivo‐Toxicity of Oligo‐Arginine Derivatives and of Novel Guanidinium‐Rich Compounds Derived from the Biopolymer Cyanophycin

ISSN:0018-019XISSN:1522-267

Transverse momentum and pseudorapidity distributions of charged hadrons in pp collisions at (s)\sqrt(s) = 0.9 and 2.36 TeV

Measurements of inclusive charged-hadron transverse-momentum and pseudorapidity distributions are presented for proton-proton collisions at sqrt(s) = 0.9 and 2.36 TeV. The data were collected with the CMS detector during the LHC commissioning in December 2009. For non-single-diffractive interactions, the average charged-hadron transverse momentum is measured to be 0.46 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 0.9 TeV and 0.50 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 2.36 TeV, for pseudorapidities between -2.4 and +2.4. At these energies, the measured pseudorapidity densities in the central region, dN(charged)/d(eta) for |eta| < 0.5, are 3.48 +/- 0.02 (stat.) +/- 0.13 (syst.) and 4.47 +/- 0.04 (stat.) +/- 0.16 (syst.), respectively. The results at 0.9 TeV are in agreement with previous measurements and confirm the expectation of near equal hadron production in p-pbar and pp collisions. The results at 2.36 TeV represent the highest-energy measurements at a particle collider to date