Identification of Two Novel Peptides That Inhibit α-Synuclein Toxicity and Aggregation.

Aggregation of α-synuclein (αSyn) into proteinaceous deposits is a pathological hallmark of a range of neurodegenerative diseases including Parkinson's disease (PD). Numerous lines of evidence indicate that the accumulation of toxic oligomeric and prefibrillar αSyn species may underpin the cellular toxicity and spread of pathology between cells. Therefore, aggregation of αSyn is considered a priority target for drug development, as aggregation inhibitors are expected to reduce αSyn toxicity and serve as therapeutic agents. Here, we used the budding yeast S. cerevisiae as a platform for the identification of short peptides that inhibit αSyn aggregation and toxicity. A library consisting of approximately one million peptide variants was utilized in two high-throughput screening approaches for isolation of library representatives that reduce αSyn-associated toxicity and aggregation. Seven peptides were isolated that were able to suppress specifically αSyn toxicity and aggregation in living cells. Expression of the peptides in yeast reduced the accumulation of αSyn-induced reactive oxygen species and increased cell viability. Next, the peptides were chemically synthesized and probed for their ability to modulate αSyn aggregation in vitro. Two synthetic peptides, K84s and K102s, of 25 and 19 amino acids, respectively, significantly inhibited αSyn oligomerization and aggregation at sub-stoichiometric molar ratios. Importantly, K84s reduced αSyn aggregation in human cells. These peptides represent promising αSyn aggregation antagonists for the development of future therapeutic interventions.

Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts.

The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aoscoi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.

CC-type glutaredoxins recruit the transcriptional co-repressor TOPLESS to TGA-dependent target promoters in Arabidopsis thaliana.

Glutaredoxins (GRXs) are small proteins which bind glutathione to either reduce disulfide bonds or to coordinate iron sulfur clusters. Whereas these well-established functions are associated with ubiquitously occurring GRXs that encode variants of a CPYC or a CGFS motif in the active center, land plants also possess CCxC/S-type GRXs (named ROXYs in Arabidopsis thaliana) for which the biochemical functions are yet unknown. ROXYs and CC-type GRXs from rice and maize physically and genetically interact with bZIP transcription factors of the TGA family to control developmental and stress-associated processes. Here we demonstrate that ROXYs interact with transcriptional co-repressors of the TOPLESS (TPL) family which are related to Tup1 in fungi and Groucho/TLE in animals. In ROXYs, the functionally important conserved A(L/I)W(L/V) motif at the very C terminus mediates the interaction with TPL. A ternary TGA2/ROXY19/TPL complex is formed when all three proteins are co-expressed in yeast. Loss-of-function evidence for the role of TPL in ROXY19-mediated repression was hampered by the redundancy of the five members of the TPL gene family and developmental defects of higher order tpl mutants. As an alternative strategy, we ectopically expressed known TPL-interacting proteins in order to out-compete the amount of available TPL in transiently transformed protoplasts. Indeed, ROXY19-mediated transcriptional repression was strongly alleviated by this approach. Our data suggest a yet unrecognized function of GRXs acting as adapter proteins for the assembly of transcriptional repressor complexes on TGA-regulated target promoters.

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis.

The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro)anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and transparent TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, Triptychon (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes.

Mutual control of intracellular localisation of the patterning proteins AtMYC1, GL1 and TRY/CPC in Arabidopsis.

Trichome and root hair patterning is governed by a gene regulatory network involving TTG1 and several homologous MYB and bHLH proteins. The bHLH proteins GL3 and EGL3 are core components that serve as a regulatory platform for the activation of downstream genes. In this study we show that a homologue of GL3 and EGL3, AtMYC1, can regulate the intracellular localisation of GL1 and TRY. AtMYC1 protein is predominantly localised in the cytoplasm and can relocate GL1 from the nucleus into the cytoplasm. Conversely, AtMYC1 can be recruited into the nucleus by TRY and CPC, concomitant with a strong accumulation of TRY and CPC in the nucleus. When AtMYC1 is targeted to the nucleus or cytoplasm by nuclear localisation or export signals (NLS or NES), respectively, the intracellular localisation of GL1 and TRY also changes accordingly. The biological significance of this intracellular localisation is suggested by the finding that the efficiency of rescue of trichome number is significantly altered in NES and NLS fusions as compared with wild-type AtMYC1. Genetic analysis of mutants and overexpression lines supports the hypothesis that AtMYC1 represses the activity of TRY and CPC.

MIDGET connects COP1-dependent development with endoreduplication in Arabidopsis thaliana.

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post-germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub-nuclear accumulation of COP1. The MID protein is not degraded in a COP1-dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1-dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1-dependent development with the regulation of endoreduplication.

Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis.

Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

The heterotrimeric G-protein complex is minimally composed of Gα, Gß, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

Trichome development in Arabidopsis.

Arabidopsis trichomes are giant single epidermal cells that are easily accessible for genetic, genomic and cell-biological analysis. They have therefore become a convenient model system to study developmental and physiological processes. Trichome studies are greatly facilitated by methods specifically applicable for this particular cell type. In addition, it is very important to use conventions and definitions that have been developed to make studies comparable and capture the relevant aspects. This chapter will highlight these two aspects of trichome analysis.

A role for ABIL3 in plant cell morphogenesis.

Actin nucleation facilitated by the ARP2/3 complex plays a central role in plant cell shape development. The molecular characterization of the distorted class of trichome mutants has recently revealed the SCAR/WAVE complex as an essential upstream activator of ARP2/3 function in plants. The SCAR/WAVE complex is conserved from animals to plants and, generally, is composed of the five subunits SCAR/WAVE, PIR121, NAP125, BRICK and ABI. In plants, four of the five subunits have been shown to participate in trichome and pavement morphogenesis. Plant ABI-like proteins (ABIL), however, which constitute a small four-member protein family in Arabidopsis thaliana, have not been characterized functionally, so far. Here we demonstrate that microRNA knock-down of the ABIL3 gene leads to a distorted trichome phenotype reminiscent of ARP2/3 mutant phenotypes and consistent with a crucial role of the ABIL3 protein in an ARP2/3-activating SCAR/WAVE complex. In contrast to ARP2/3 mutants, however, the ABIL3 knock-down stimulated cell elongation in the root, indicating distinct functions of the ABIL3 protein in different tissues. Furthermore, we provide evidence that ABIL3 associates with microtubules in vivo, opening up the intriguing possibility that ABI-like proteins have a function in linking SCAR/WAVE-dependent actin nucleation with organization of the microtubule cytoskeleton.

Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling.

Mitogen-activated protein kinase (MAPK)-mediated responses are in part regulated by the repertoire of MAPK substrates, which is still poorly elucidated in plants. Here, the in vivo enzyme-substrate interaction of the Arabidopsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is shown by fluorescence resonance energy transfer. The interaction was rapidly lost in response to flagellin-derived flg22 peptide. This complex disruption requires not only MPK6 activity, which also affects ERF104 stability via phosphorylation, but also ethylene signaling. The latter points to a novel role of ethylene in substrate release, presumably allowing the liberated ERF104 to access target genes. Microarray data show enrichment of GCC motifs in the promoters of ERF104-up-regulated genes, many of which are stress related. ERF104 is a vital regulator of basal immunity, as altered expression in both erf104 and overexpressors led to more growth inhibition by flg22 and enhanced susceptibility to a non-adapted bacterial pathogen.

Yeast two-hybrid assay to identify host-virus interactions.

The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way the interactions that exist between virus and plant. A variety of methods are now available to isolate and study interacting proteins, however, the yeast two-hybrid (Y2H) assay system, which was one of the earliest mass analysis methods to be developed [Nature 340:245-246, 1989] remains one of the most popular and amenable approaches in current use. The Y2H method works by expressing two candidate interacting proteins together in the yeast cell. The (bait and prey) proteins under study are fused either to a promoter-specific DNA-binding domain or to a transcription activation domain. Interaction in the yeast nucleus between the bait and prey proteins brings the transcription activation and DNA-binding domains together so that they can initiate expression of a reporter gene. The reporter may be nonselective, such as the beta-galactosidase (LacZ) protein, or be selective by complementing a chromosomal mutation in a metabolic pathway for, for example, leucine or histidine biosynthesis. Individual bait proteins can be screened for interaction against a library of prey proteins, with any yeast colonies that grow on selective plates containing potential interacting partners. Using the Y2H system, a number of plant proteins interacting with viral proteins have been identified, recently, increasing our knowledge of the molecular basis of viral infection and host defense mechanisms.

Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis.

Interactions between plants and fungal pathogens require a complex interplay at the plant-fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens.

MIDGET unravels functions of the Arabidopsis topoisomerase VI complex in DNA endoreduplication, chromatin condensation, and transcriptional silencing.

The plant homologs of the archaeal DNA topoisomerase VI complex are required for the progression of endoreduplication cycles. Here, we describe the identification of MIDGET (MID) as a novel component of topoisomerase VI. We show that mid mutants show the same phenotype as rhl1, rhl2, and top6B mutants and that MID protein physically interacts with RHL1. The phenotypic analysis revealed new phenotypes, indicating that topoisomerase VI is involved in chromatin organization and transcriptional silencing. In addition, genetic evidence is provided suggesting that the ATR-dependent DNA damage repair checkpoint is activated in mid mutants, and CYCB1;1 is ectopically activated. Finally, we demonstrate that overexpression of CYCB1;2 can rescue the endoreduplication defects in mid mutants, suggesting that in mid mutants, a specific checkpoint is activated preventing further progression of endoreduplication cycles.

The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis.

The actin-nucleating ARP2-ARP3 complex controls cell shape in plants in many different cell types. Its activity is controlled by a multimeric complex containing BRK1 (also known as HSPC300), NAP1, SRA1, ABI and SCAR/WAVE. In this study, we focus on the function of the five putative SCAR homologues in Arabidopsis and we provide biochemical evidence that AtSCAR2 can activate the ARP2-ARP3 complex in vitro. Among the single mutants, mutations in only AtSCAR2 result in a subtle or weak phenotype similar to ARP2, ARP3 and other ;distorted' mutants. Double-mutant analysis revealed a redundancy with AtSCAR4. Systematic application of the yeast two-hybrid system and Bimolecular Fluorescence Complementation (BiFC) revealed a complex protein-interaction network between the ARP2-ARP3 complex and its genetically defined regulators. In addition to protein interactions known in other systems, we identified several new interactions, suggesting that SPIKE1 may be an integral component of the SCAR/WAVE complex and that SCAR proteins in plants might act as direct effectors of ROP GTPases.

Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity.

The P19 protein of Tomato bushy stunt virus is a potent suppressor of RNA silencing and, depending on the host species, is required for short- and long-distance virus movement and symptom production. P19 interacts with plant ALY proteins and relocalizes a subset of these proteins from the nucleus to the cytoplasm. Here we showed that coexpression by agroinfiltration in Nicotiana benthamiana of P19 and the subset of ALY proteins that are not relocalized from the nucleus interfered with the ability of P19 to suppress RNA silencing. We demonstrated that this interference correlates with the relocation of P19 from the cytoplasm into the nucleus, and by constructing and analyzing chimeric ALY genes, we showed that the C-terminal part of the central, RNA recognition motif of ALY is responsible for interaction with P19, relocalization or nonrelocalization of ALY, and inhibition of silencing suppression by P19. We studied the interaction of ALY and P19 by using the technique of bimolecular fluorescence complementation to show that these proteins associate physically in the nucleus but not detectably in the cytoplasm, and we present a model to explain the dynamics of this interaction.

An improved mRFP1 adds red to bimolecular fluorescence complementation.

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.

Protein interaction networks in plants.

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. With the development of high-throughput technologies of both the yeast two-hybrid system and tandem mass spectrometry, genome-wide protein-linkage mapping has become a major objective in post-genomic research. While at least partial "interactome" networks of several model organisms are already available, in the plant field, progress in this respect is slow. However, even with comprehensive protein interaction data still missing, substantial recent advance in the graph-theoretical functional interpretation of complex network architectures might pave the way for novel approaches in plant research. This article reviews current progress and discussions in network biology. Emphasis is put on the question of what can be learned about protein functions and cellular processes by studying the topology of complex protein interaction networks and the evolutionary mechanisms underlying their development. Particularly the intermediate and local levels of network organization--the modules, motifs and cliques--are increasingly recognized as the operational units of biological functions. As demonstrated by some recent results from systematic analyses of plant protein families, protein interaction networks promise to be a valuable tool for a molecular understanding of functional specificities and for identifying novel regulatory components and pathways.

The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis.

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of D-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Galpha interaction partner that functions downstream of the plasma membrane-delimited heterotrimeric G-protein (GPA1) in a D-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by Förster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous D-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high D-glucose. Additionally, THF1 levels are rapidly degraded by D-glucose but not L-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.

Plant GTPases: regulation of morphogenesis by ROPs and ROS.

Polarized cell growth in plants is controlled by Rho-like small GTPases (ROPs), not only through the canonical WAVE/Arp2/3 pathway, but also through newly defined plant-specific pathways involving the regulated release of reactive oxygen species (ROS).