Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils

Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.The tripeptide glutathione (GSH)² is abundant in the cell. It plays an important role as a reducing agent in vivo, such as in endogenous free radical scavenging, reversible protein S-glutathionylation, and the reduction of the active sites of enzymes. One major class of enzyme that uses GSH as a reductant is glutaredoxin (GRX), which is a small protein involved in reduction of ribonucleotide reductase for the formation of deoxyribonucleotides for DNA synthesis (1), reduction of 3′-phosphoadenylylsulfate reductase (2) for generation of sulfite, signal transduction, and protection against oxidative stress (3). GRXs are ubiquitous thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily (4). GRXs also show dehydroascorbic acid (DHA) reductase (DHAR) activity (5). Yeast Saccharomyces cerevisiae has at least seven GRXs, which can be divided into two classes according to the number of cysteines in their active site motif: dithiol GRXs with the active site motif CXXC and monothiol GRXs with the motif CXXS (6–9). The dithiol GRXs catalyze protein disulfide reduction using a dithiol mechanism for which both the active site cysteines are essential. On the other hand, both the dithiol and monothiol GRXs can catalyze the reduction of GSH-protein mixed disulfides using a monothiol mechanism that only requires the N-terminal active site cysteine. This reaction and mechanism is important for reversible protein glutathionylation in redox signaling and oxidative stress (10).Glutathione S-transferases (GSTs) are a large versatile family of enzymes with multiple functions, particularly associated with cellular detoxification (11). In terms of overall structure, they belong to the thioredoxin superfamily, like GRX (4). In general, GSTs catalyze the conjugation of reduced GSH to hydrophobic substrates containing an electrophilic atom. In addition, GSTs bind a broad spectrum of ligands and show many other functions. For example, some GSTs show overlapping functions with glutathione-dependent peroxidases (GPxs), which use GSH to reduce hydrogen peroxide and/or organic hydroperoxides and thus are responsible for protection against both endogenous and exogenous oxidant toxicity (11). Interestingly Omega class and Beta class GSTs (such as Escherichia coli GST (EGST)) possess typical GRX activity toward widely used substrates, such as 2-hydroxyethyl disulfide (HEDS) (12–16). These GSTs have an active site cysteine, which is indispensable for GRX activity but not GST activity.The yeast prion protein Ure2 is composed of a disordered protease-sensitive N-terminal prion domain and a compact globular C-terminal domain, which shows high structural similarity to EGST (17). The C-terminal domain of Ure2 can be further structurally divided into two subdomains, the all-α-helix subdomain and the thioredoxin fold subdomain, which shows high structural homology to GRX. Ure2 is involved in the regulation of nitrogen metabolism and resistance to heavy metal ion toxicity (especially cadmium) and oxidative stress in S. cerevisiae (18, 19). In addition, Ure2 shows GPx activity toward both hydrogen peroxide and organic hydroperoxides such as cumene hydroperoxide and tert-butyl hydroperoxide (20). The discovery of the GPx activity of Ure2 (20) provides an explanation for its ability to protect yeast cells from oxidant toxicity (18). However, the reason that ure2Δ yeast cells are hypersensitive to cadmium remains unclear. In general, cadmium ions have a drastic effect on yeast cell growth, and the reasons are complicated. One possible reason for cadmium ion toxicity is that thioltransferases or GRXs can be inhibited by direct binding of cadmium to the two essential cysteine residues present in the thioltransferase active site (21). The inhibition of GRXs leads to complex effects on cell growth. Therefore, we used an in vitro assay to provide a system that allows detailed analysis of the activity of Ure2 and its relationship to that of GRX enzymes. Characterization of the allosteric behavior of the GRX activity of Ure2 revealed that Ure2 forms an active dimer within fibrils. In addition to providing information about the molecular structure of Ure2 fibrils, this also has implications for the molecular mechanism of Ure2 prion formation.     

Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant Concentration

Prion protein is known to have the ability to adopt a pathogenic conformation, which seems to be the basis for protein-only infectivity. The infectivity is based on self-replication of this pathogenic prion structure. One of possible mechanisms for such replication is the elongation of amyloid-like fibrils.We measured elongation kinetics and thermodynamics of mouse prion amyloid-like fibrils at different guanidine hydrochloride (GuHCl) concentrations. Our data show that both increases in temperature and GuHCl concentration help unfold monomeric protein and thus accelerate elongation. Once the monomers are unfolded, further increases in temperature raise the rate of elongation, whereas the addition of GuHCl decreases it.We demonstrated a possible way to determine different activation energies of amyloid-like fibril elongation by using folded and unfolded protein molecules. This approach separates thermodynamic data for fibril-assisted monomer unfolding and for refolding and formation of amyloid-like structure.     

Soy Peptides Enhance Heterologous Membrane Protein Productivity during the Exponential Growth Phase of Saccharomyces cerevisiae

In this study, the production of eight G protein-coupled receptors by Saccharomyces cerevisiae was compared using two types of media, one of which contained soy peptides and the other free amino acids. Yeast cell growth improved in the medium with soy peptides, and the expression levels of six of the receptors increased during the exponential phase by an average of 2.3-fold as against the free amino acid-based medium. The enhancement of protein expression by soy peptides can be explained by alleviation of metabolite stress due to amino acid source depletion caused by heterologous protein expression.     

Functionally Relevant Domains of the Prion Protein Identified In Vivo

The prion consists essentially of PrP^Sc, a misfolded and aggregated conformer of the cellular protein PrP^C. Whereas PrP^C deficient mice are clinically healthy, expression of PrP^C variants lacking its central domain (PrP_ΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrP^C (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrP_ΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrP_ΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrP_ΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants.     

The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD⁺ plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.Tyrosine serves as a precursor for the synthesis of proteins and secondary metabolites such as quinones (1-3), alkaloids (4), flavonoids (5), and phenolic compounds (5, 6). In prokaryotes and plants, these compounds are important for viability and normal development (7).The TyrA protein family consists of dehydrogenase homologues that are dedicated to the biosynthesis of l-tyrosine. These enzymes participate in two independent metabolic branches that result in the conversion of prephenate to l-tyrosine, namely the arogenate route and the 4-hydroxyphenylpyruvate (HPP)³ routes. Although both of these pathways utilize a common precursor and converge to produce a common end-product, they differ in the sequential order of enzymatic steps. Through the HPP route, prephenate is first decarboxylated by prephenate dehydrogenase (PD) to yield HPP, which is subsequently transaminated to l-tyrosine via a TyrB homologue (8). Alternatively, through the arogenate route, prephenate is first transaminated to l-arogenate by prephenate aminotransferase and then decarboxylated by arogenate dehydrogenase (AD) to yield l-tyrosine (9-11) (see Fig. 1A).Open in a separate windowFIGURE 1.A, metabolic routes from chorismate leading to the synthesis of l-tyrosine and l-phenylalanine. In the arogenate, 4-hydroxyphenylpyruvate, or phenylpyruvate route, prephenate and arogenate are branch point intermediates in both l-tyrosine and l-phenylalanine biosynthesis. Prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate with NAD⁺ to produce hydroxyphenylpyruvate, NADH, and CO₂ (40). B, a comparison of the chemical structure of the three ligands, HPP, HPpropionate, and tyrosine, used in the crystallization of A. aeolicus prephenate dehydrogenase. These ligands all have an -OH at the C4 position and a propionyl side chain at the C1 position of the ring.There are three classes of TyrA enzymes that catalyze the oxidative decarboxylation reactions in these two pathways. The enzymes are distinguished by the affinity for cyclohexadienyl substrates. PD and AD accept prephenate or l-arogenate, respectively, whereas the cyclohexadienyl dehydrogenases can catalyze the reaction using either substrate (12).To ensure efficient metabolite distribution of the pathway intermediates, TyrA enzymes are highly regulated by various control mechanisms, including feedback inhibition, and genetic regulation by the Tyr operon (13-16). In some cases, l-tyrosine competes directly with substrate, be it prephenate or l-arogenate for the active site of arogenate or cyclohexadienyl dehydrogenases (14, 17-19). The product HPP can also serve as an efficient competitive inhibitor with respect to prephenate (20). Additionally, at the protein level PDs are only shown to be regulated at distinct allosteric sites or domains to modulate their activity. For example, the results of kinetic studies on the bifunctional Escherichia coli chorismate mutase-prephenate dehydrogenase (CM-PD) have indicated that this enzyme likely possesses a distinct allosteric site for binding tyrosine (21). In contrast, the Bacillus subtilis PD is the only enzyme reported to be competitively inhibited by HPP and l-tyrosine but is also noncompetitively inhibited by l-phenylalanine and l-tryptophan (12, 22). Additional regulatory control is thought to originate through a C-terminal aspartate kinase-CM-TyrA domain of the B. subtilis PD (23).Biochemical analyses of PD from E. coli CM-PD have provided a framework for understanding the molecular mechanism of the TyrA enzymes. The E. coli PD-catalyzed reaction proceeds though a rapid equilibrium, random kinetic mechanism with catalysis as the rate-limiting step (24). Additionally, studies of the pH dependence of the kinetic parameters V and V/K indicate that a deprotonated group facilitates hydride transfer from prephenate to NAD⁺ by polarizing the 4-hydroxyl group of prephenate, whereas a protonated residue is required for binding prephenate to the enzyme·NAD⁺ complex (25). The conserved residues His-197 and Arg-294 have been identified through extensive mutagenesis studies to fulfill these two roles (26, 27). Further analyses of the activities of wild-type protein and site-directed variants in the presence of a series of inhibitory substrate analogues support the idea that Arg-294 binds prephenate through the ring carboxylate (26).The structures of AD from Synechocystis sp. and PD from Aquifex aeolicus (both in complex with NAD⁺) have been reported by Legrand et al. (28) and by our group (29), respectively. Analyses of these structures have provided structural information on the conserved histidine and arginine residues. The structure A. aeolicus PD has also led to the identification of other active site residues that may play a role in enzyme catalysis, most notably Ser-126, which we propose facilitates catalysis by orienting the catalytic histidine and the nicotinamide moiety of NAD⁺ into their catalytically efficient conformations. Ambiguities can arise from examination of the binary complexes, because prephenate has only been modeled in the active site. For example, analysis of the AD structure by Legrand et al. (28) places Arg-217 (equivalent to Arg-294 in E. coli and Arg-250 in A. aeolicus) too far from the active site to play a role in prephenate binding. Thus, the full complement of interactions between prephenate and TyrA proteins are still largely unknown, as are the interactions of the enzymes with l-tyrosine.To further investigate the importance of residues involved in ligand binding, specificity, and catalysis, we have carried out co-crystallization studies of A. aeolicus PD with NAD⁺ and prephenate, with NAD⁺ and 4-hydroxyphenylpropionate (HPpropionate), a product analogue, and with NAD⁺ and l-tyrosine. Accordingly, this study provides the first direct evidence that l-tyrosine binds to the active site of a prephenate dehydrogenase. We have investigated the role of Ser-126, His-147, His-217, and Arg-250 through the kinetic analysis of site-directed mutants and structural analysis of the co-crystal complexes. To understand the role of active site residues in substrate selectivity, comparative structural analysis of AD and PD was also conducted. The current study provides a basis for understanding the mechanism of substrate selectivity between the different classes of TyrA enzymes and details how A. aeolicus PD can accept prephenate as substrate and l-tyrosine as a competitive inhibitor.     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

Mitotic Phosphorylation of Golgi Reassembly Stacking Protein 55 by Mitogen-activated Protein Kinase ERK2

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.     

MazF-induced Growth Inhibition and Persister Generation in Escherichia coli

Toxin-antitoxin systems are ubiquitous in nature and present on the chromosomes of both bacteria and archaea. MazEF is a type II toxin-antitoxin system present on the chromosome of Escherichia coli and other bacteria. Whether MazEF is involved in programmed cell death or reversible growth inhibition and bacterial persistence is a matter of debate. In the present work the role of MazF in bacterial physiology was studied by using an inactive, active-site mutant of MazF, E24A, to activate WT MazF expression from its own promoter. The ectopic expression of E24A MazF in a strain containing WT mazEF resulted in reversible growth arrest. Normal growth resumed on inhibiting the expression of E24A MazF. MazF-mediated growth arrest resulted in an increase in survival of bacterial cells during antibiotic stress. This was studied by activation of mazEF either by overexpression of an inactive, active-site mutant or pre-exposure to a sublethal dose of antibiotic. The MazF-mediated persistence phenotype was found to be independent of RecA and dependent on the presence of the ClpP and Lon proteases. This study confirms the role of MazEF in reversible growth inhibition and persistence.     

Fucosyltransferase and the Biosynthesis of Storage and Structural Xyloglucan in Developing Nasturtium Fruits

Young, developing fruits of nasturtium (Tropaeolum majus L.) accumulate large deposits of nonfucosylated xyloglucan (XG) in periplasmic spaces of cotyledon cells. This “storage” XG can be fucosylated by a nasturtium transferase in vitro, but this does not happen in vivo, even as a transitory signal for secretion. The only XG that is clearly fucosylated in these fruits is the structural fraction (approximately 1% total) that is bound to cellulose in growing primary walls. The two fucosylated subunits that are formed in vitro are identical to those found in structural XG in vivo. The yield of XG-fucosyltransferase activity from membrane fractions is highest per unit fresh weight in the youngest fruits, especially in dissected cotyledons, but declines when storage XG is forming. A block appears to develop in the secretory machinery of young cotyledon cells between sites that galactosylate and those that fucosylate nascent XG. After extensive galactosylation, XG traffic is diverted to the periplasm without fucosylation. The primary walls buried beneath accretions of storage XG eventually swell and lose cohesion, probably because they continue to extend without incorporating components such as fucosylated XG that are needed to maintain wall integrity.     

Prokaryotic Ubiquitin-Like ThiS Fusion Enhances the Heterologous Protein Overexpression and Aggregation in Escherichia coli

Fusion tags are commonly employed to enhance target protein expression, improve their folding and solubility, and reduce protein degradation in expression of recombinant proteins. Ubiquitin (Ub) and SUMO are highly conserved small proteins in eukaryotes, and frequently used as fusion tags in prokaryotic expression. ThiS, a smaller sulfur-carrier protein involved in thiamin synthesis, is conserved among most prokaryotic species. The structural similarity between ThiS and Ub provoked us into expecting that the former could be used as a fusion tag. Hence, ThiS was fused to insulin A and B chains, murine Ribonuclease Inhibitor (mRI) and EGFP, respectively. When induced in Escherichia coli, ThiS-fused insulin A and B chains were overexpressed in inclusion bodies, and to higher levels in comparison to the same proteins fused with Ub. On the contrast, ThiS fusion of mRI, an unstable protein, resulted in enhanced degradation that was not alleviated in protease-deficient strains. While the degradation of Ub- and SUMO-fused mRI was less and seemed protease-dependent. Enhanced degradation of mRI did not occur for the fusions with half-molecules of ThiS. When ThiS-tag was fused to the C-terminus of EGFP, higher expression, predominantly in inclusion bodies, was observed again. It was further found that ThiS fusion of EGFP significantly retarded its refolding process. These results indicated that prokaryotic ThiS is able to promote the expression of target proteins in E. coli, but enhanced degradation may occur in case of unstable targets. Unlike eukaryotic Ub-based tags usually increase the solubility and folding of proteins, ThiS fusion enhances the expression by augmenting the formation of inclusion bodies, probably through retardation of the folding of target proteins.     

Analysis of Protein Phosphatase-1 and Aurora Protein Kinase Suppressors Reveals New Aspects of Regulatory Protein Function in Saccharomyces cerevisiae

Protein phosphatase-1 (PP1) controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates.     

Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrP^C molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrP^C is pre-incubated with poly(A) RNA and PrP^Sc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP^Sc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrP^Sc molecules, most likely through an epitope containing residue 172.     

Quantitative Analysis of the Mitochondrial and Plastid Proteomes of the Moss Physcomitrella patens Reveals Protein Macrocompartmentation and Microcompartmentation

Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context.Endosymbiosis has enabled and shaped eukaryotic evolution. The engulfment of an ancestral α-proteobacterium by a presumably archaebacterial host cell stands at the origin of mitochondrial and eukaryotic evolution over 1.5 billion years ago (Dyall et al., 2004). In plants, the subsequent uptake of a photosynthetic bacterium between 1.5 and 1.2 billion years ago led to the formation of chloroplasts (Dyall et al., 2004). Plants thereby evolved by the integration of three distinct genetic compartments. After the establishment of endosymbiosis, genes were transferred to a great extent, mainly from mitochondria and plastids to the nucleus (Bock and Timmis, 2008), necessitating an orchestrated flux of information in the form of proteins and metabolites between the compartments of eukaryotic cells to ensure homeostasis, growth, and development. This communication between organelles is facilitated by physical interactions (Kornmann et al., 2009), control of protein import (Ling et al., 2012), and retrograde signaling (Nargund et al., 2012). During radiation and diversification, especially of land plants, nuclear genomes substantially changed due to endosymbiotic and horizontal (Yue et al., 2012) gene transfer, genome duplication, and gene gain and loss (Duarte et al., 2006; Lang et al., 2010; Martin, 2010), obtruding the question of whether these phenomena are linked to alterations in metabolic pathway partitioning between organelles. Retained paralogs can either introduce a new function (neofunctionalization) or reconstitute existing functions (subfunctionalization; Duarte et al., 2006), for example by distinct spatiotemporal expression profiles or distinct subcellular localizations, resulting in the modulation or introduction of metabolic functions in the respective cellular compartments. Moreover, proteins can localize to several subcellular compartments, a phenomenon called dual or multiple targeting (Yogev and Pines, 2011; Xu et al., 2013). Consequently, many eukaroytic metabolic pathways, as well as the plastid and mitochondrial proteomes, are constituted of a mosaic of proteins of diverse evolutionary origins (Szklarczyk and Huynen, 2010), and evolution has shaped variable organellar functionalities across taxa. To date, the evolution and variability of postendosymbiotic metabolic partitioning is largely not characterized on a high-throughput level. So far, large-scale mitochondrial proteome data sets are only available for the green alga Chlamydomonas reinhardtii (Atteia et al., 2009), rice (Oryza sativa; Huang et al., 2009), and the model flowering plant Arabidopsis (Arabidopsis thaliana; Millar et al., 2001; Heazlewood et al., 2004), whereas plastid proteomics in plants is on an advanced level and covers more species (Polyakov et al., 2010; van Wijk and Baginsky, 2011).While higher plants diversified relatively recently but massively, simple moss plants can be traced back 330 million years (Hubers and Kerp, 2012), identifying them as prime candidates for an evolutionary view of organellar proteomes and organelle biology at a genome-wide scale. In contrast to specialized flowering plants, mosses are generalists with few tissues, high metabolic variability, and ancestral features such as high abiotic stress tolerance (Frank et al., 2007) and few plastid types (Cove, 2005).By integrating quantitative proteomics, multivariate analysis, metabolic pathway maps, phylogenomics, and localization with fluorescent proteins, we reliably characterize subcellular proteomes and gene family diversification. Key characteristics of postendosymbiotic organellar proteome evolution are identified by cross-species comparative analysis. In support of our high-throughput analyses, we conduct single-protein analyses and identify proteins that mark microcompartments within organelles and localize to dynamic contact sites between organelles. These proteins may facilitate the exchange of proteins and metabolites, while others influence the dynamics of individual chloroplasts and mitochondria. This study characterizes the mitochondrial and plastid proteomes of moss and reveals the heterogeneity of organelles within a single cell.     

Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Derlin2 Protein Facilitates HRD1-mediated Retro-translocation of Sonic Hedgehog at the Endoplasmic Reticulum

Endoplasmic reticulum-associated degradation (ERAD) is an important system that eliminates misfolded proteins from the ER. Three derlins have been implicated in this process, but their precise function remains unknown. In this study, we report that although both derlin1 and derlin2 are capable of binding the ERAD-specific ubiquitin ligase HRD1, they associate with the HRD1-containing complex with different affinities. Accordingly, these derlins have nonredundant functions in ERAD with derlin2 being an essential functional partner for HRD1-mediated ERAD of SHH and NHK. We show that derlin2, but not derlin1 or derlin3, is required for ERAD of both glycosylated and nonglycosylated SHH, as well as NHK. Derlin2 appears to act at a post-targeting step for HRD1-dependent retro-translocation. Without derlin2, the assembly of HRD1 into a functional retro-translocation homo-oligomer proceeds normally, and substrate targeting to the HRD1 complex also occurs. However, the ERAD substrate SHH-C is largely trapped inside the ER lumen. These observations raise the possibility that derlin2 may regulate the movement of substrates through the HRD1-containing retro-translocon. Our study is the first to report that derlin2 functions with HRD1 in ERAD of certain substrates independent of their glycosylation status. The mammalian ERAD system may require multiple derlins that each functions with a distinct E3 partner to eliminate a specific subset of substrates. This is different from the model in Saccharomyces cerevisiae, in which Hrd1p alone is sufficient for retro-translocation.     

Crystal Structure of the cGMP-dependent Protein Kinase II Leucine Zipper and Rab11b Protein Complex Reveals Molecular Details of G-kinase-specific Interactions

cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40–83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the β1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iβ LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.     

Development of the Structural Core and of Conformational Heterogeneity during the Conversion of Oligomers of the Mouse Prion Protein to Worm-like Amyloid Fibrils

Understanding how structure develops during the course of amyloid fibril formation by the prion protein is important for understanding prion diseases. Determining how conformational heterogeneity manifests itself in the fibrillar and pre-fibrillar amyloid aggregates is critical for understanding prion strain phenotypes. In this study, the formation of worm-like amyloid fibrils by the mouse prion protein has been characterized structurally by hydrogen-deuterium exchange coupled to mass spectrometry. The structural cores of these fibrils and of the oligomer on the direct pathway of amyloid fibril formation have been defined, showing how structure develops during fibril formation. The structural core of the oligomer not on the direct pathway has also been defined, allowing the delineation of the structural features that make this off-pathway oligomer incompetent to directly form fibrils. Sequence segments that exhibit multiple local conformations in the three amyloid aggregates have been identified, and the development of structural heterogeneity during fibril formation has been characterized. It is shown that conformational heterogeneity is not restricted to only the C-terminal domain region, which forms the structural core of the aggregates; it manifests itself in the N-terminal domain of the protein as well. Importantly, all three amyloid aggregates are shown to be capable of disrupting lipid membrane structure, pointing to a mechanism by which they may be toxic.     

Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWP_LRR) and a C-terminal conserved Cys-rich region (CWP_CRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (∼400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (∼1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1_LRR. In contrast, neither MBP alone nor MBP fused to CWP1_CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.