In silico analysis of the cyclophilin repertoire of apicomplexan parasites

<p>Abstract</p> <p>Background</p> <p>Cyclophilins (Cyps) are peptidyl <it>cis/trans </it>isomerases implicated in diverse processes such as protein folding, signal transduction, and RNA processing. They are also candidate drug targets, in particular for the immunosuppressant cyclosporine A. In addition, cyclosporine is known to exhibit anti-parasitic effects on a wide range of organisms including several apicomplexa. In order to obtain new non-immunosuppressive drugs targeting apicomplexan cyclophilins, a profound knowledge of the cyclophilin repertoire of this phylum would be necessary.</p> <p>Results</p> <p>BLAST and maximum likelihood analyses identified 16 different cyclophilin subfamilies within the genomes of <it>Cryptosporidium hominis</it>, <it>Toxoplasma gondii</it>, <it>Plasmodium falciparum</it>, <it>Theileria annulata</it>, <it>Theileria parva</it>, and <it>Babesia bovis</it>. In addition to good statistical support from the phylogenetic analysis, these subfamilies are also confirmed by comparison of cyclophilin domain architecture. Within an individual genome, the number of different Cyp genes that could be deduced varies between 7–9 for Cryptosporidia and 14 for <it>T. gondii</it>. Many of the putative apicomplexan cyclophilins are predicted to be nuclear proteins, most of them presumably involved in RNA processing.</p> <p>Conclusion</p> <p>The genomes of apicomplexa harbor a cyclophilin repertoire that is at least as complex as that of most fungi. The identification of Cyp subfamilies that are specific for lower eukaryotes, apicomplexa, or even the genus Plasmodium is of particular interest since these subfamilies are not present in host cells and might therefore represent attractive drug targets.</p

Homo-dimerization and ligand binding by the leucine-rich repeat domain at RHG1/RFS2 underlying resistance to two soybean pathogens

BACKGROUND: The protein encoded by GmRLK18-1 (Glyma_18_02680 on chromosome 18) was a receptor like kinase (RLK) encoded within the soybean (Glycine max L. Merr.) Rhg1/Rfs2 locus. The locus underlies resistance to the soybean cyst nematode (SCN) Heterodera glycines (I.) and causal agent of sudden death syndrome (SDS) Fusarium virguliforme (Aoki). Previously the leucine rich repeat (LRR) domain was expressed in Escherichia coli. RESULTS: The aims here were to evaluate the LRRs ability to; homo-dimerize; bind larger proteins; and bind to small peptides. Western analysis suggested homo-dimers could form after protein extraction from roots. The purified LRR domain, from residue 131–485, was seen to form a mixture of monomers and homo-dimers in vitro. Cross-linking experiments in vitro showed the H274N region was close (<11.1 A) to the highly conserved cysteine residue C196 on the second homo-dimer subunit. Binding constants of 20–142 nM for peptides found in plant and nematode secretions were found. Effects on plant phenotypes including wilting, stem bending and resistance to infection by SCN were observed when roots were treated with 50 pM of the peptides. Far-Western analyses followed by MS showed methionine synthase and cyclophilin bound strongly to the LRR domain. A second LRR from GmRLK08-1 (Glyma_08_g11350) did not show these strong interactions. CONCLUSIONS: The LRR domain of the GmRLK18-1 protein formed both a monomer and a homo-dimer. The LRR domain bound avidly to 4 different CLE peptides, a cyclophilin and a methionine synthase. The CLE peptides GmTGIF, GmCLE34, GmCLE3 and HgCLE were previously reported to be involved in root growth inhibition but here GmTGIF and HgCLE were shown to alter stem morphology and resistance to SCN. One of several models from homology and ab-initio modeling was partially validated by cross-linking. The effect of the 3 amino acid replacements present among RLK allotypes, A87V, Q115K and H274N were predicted to alter domain stability and function. Therefore, the LRR domain of GmRLK18-1 might underlie both root development and disease resistance in soybean and provide an avenue to develop new variants and ligands that might promote reduced losses to SCN

Characterization of Fsr1-Interacting Complex and Its Downstream Pathogenic Subnetwork Modules in Fusarium verticillioides

Fusarium verticillioides is an ascomycete fungus responsible for stalk and ear rots of maize. Previously, we identified a striatin-like protein Fsr1 that plays a key role in stalk rot pathogenesis. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin-interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homolog of a key mammalian STRIPAK component STRIP1/2 in F. verticillioides, FvStp1, that interacts with Fsr1 in vivo. Gene deletion analysis showed that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, that interact with the Fsr1 CC domain by yeast-two-hybrid screen. Importantly, FvCyp1, FvScp1, and FvSel1 co-localize to endomembrane structures, each having preferred localization in the cell, and they are all required for F. verticillioides virulence in stalk rot. Moreover, these proteins are necessary for proper localization of Fsr1 to endoplasmic reticulum (ER) and nuclear envelope. To further characterize genetic networks downstream of Fsr1, we performed RNA-Seq with maize B73 stalks inoculated with wild type and fsr1 mutant. We used a computationally efficient branch-out technique, along with an adopted probabilistic pathway activity inference method, to identify functional subnetwork modules likely involved in F. verticillioides virulence. We identified two putative hub genes, i.e., FvSYN1 and FvEBP1 identified from the potential virulence-associated subnetwork modules for functional validation and network robustness studies, such as gene knockout, virulence assays and qPCR studies. Our results provide evidence that FvSYN1 and FvEBP1 are important virulence genes that can infulence the expression of closely correlated genes, providing evidence that these are important hub genes of their respective subnetworks. Further characterization of FvSYN1 showed that FvSyn1 is important for regulating spore germination and hyphal morphology. Furthermore, FvSyn1 is localized to vacuoles, plasma membranes, and septa, and has been shown to play a role in the response to cell wall stressors. Motif-deletion studies showed that both N-terminal SynN domain and C-terminal SNARE domain of FvSyn1 are required for pathogenicity but dispensable for fumonisin production and sexual mating

Characterization of Fsr1-Interacting Complex and Its Downstream Pathogenic Subnetwork Modules in Fusarium verticillioides

Fusarium verticillioides is an ascomycete fungus responsible for stalk and ear rots of maize. Previously, we identified a striatin-like protein Fsr1 that plays a key role in stalk rot pathogenesis. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin-interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homolog of a key mammalian STRIPAK component STRIP1/2 in F. verticillioides, FvStp1, that interacts with Fsr1 in vivo. Gene deletion analysis showed that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, that interact with the Fsr1 CC domain by yeast-two-hybrid screen. Importantly, FvCyp1, FvScp1, and FvSel1 co-localize to endomembrane structures, each having preferred localization in the cell, and they are all required for F. verticillioides virulence in stalk rot. Moreover, these proteins are necessary for proper localization of Fsr1 to endoplasmic reticulum (ER) and nuclear envelope. To further characterize genetic networks downstream of Fsr1, we performed RNA-Seq with maize B73 stalks inoculated with wild type and fsr1 mutant. We used a computationally efficient branch-out technique, along with an adopted probabilistic pathway activity inference method, to identify functional subnetwork modules likely involved in F. verticillioides virulence. We identified two putative hub genes, i.e., FvSYN1 and FvEBP1 identified from the potential virulence-associated subnetwork modules for functional validation and network robustness studies, such as gene knockout, virulence assays and qPCR studies. Our results provide evidence that FvSYN1 and FvEBP1 are important virulence genes that can infulence the expression of closely correlated genes, providing evidence that these are important hub genes of their respective subnetworks. Further characterization of FvSYN1 showed that FvSyn1 is important for regulating spore germination and hyphal morphology. Furthermore, FvSyn1 is localized to vacuoles, plasma membranes, and septa, and has been shown to play a role in the response to cell wall stressors. Motif-deletion studies showed that both N-terminal SynN domain and C-terminal SNARE domain of FvSyn1 are required for pathogenicity but dispensable for fumonisin production and sexual mating

A structural classification of protein-protein interactions for detection of convergently evolved motifs and for prediction of protein binding sites on sequence level

BACKGROUND: A long-standing challenge in the post-genomic era of Bioinformatics is the prediction of protein-protein interactions, and ultimately the prediction of protein functions. The problem is intrinsically harder, when only amino acid sequences are available, but a solution is more universally applicable. So far, the problem of uncovering protein-protein interactions has been addressed in a variety of ways, both experimentally and computationally. MOTIVATION: The central problem is: How can protein complexes with solved threedimensional structure be utilized to identify and classify protein binding sites and how can knowledge be inferred from this classification such that protein interactions can be predicted for proteins without solved structure? The underlying hypothesis is that protein binding sites are often restricted to a small number of residues, which additionally often are well-conserved in order to maintain an interaction. Therefore, the signal-to-noise ratio in binding sites is expected to be higher than in other parts of the surface. This enables binding site detection in unknown proteins, when homology based annotation transfer fails. APPROACH: The problem is addressed by first investigating how geometrical aspects of domain-domain associations can lead to a rigorous structural classification of the multitude of protein interface types. The interface types are explored with respect to two aspects: First, how do interface types with one-sided homology reveal convergently evolved motifs? Second, how can sequential descriptors for local structural features be derived from the interface type classification? Then, the use of sequential representations for binding sites in order to predict protein interactions is investigated. The underlying algorithms are based on machine learning techniques, in particular Hidden Markov Models. RESULTS: This work includes a novel approach to a comprehensive geometrical classification of domain interfaces. Alternative structural domain associations are found for 40% of all family-family interactions. Evaluation of the classification algorithm on a hand-curated set of interfaces yielded a precision of 83% and a recall of 95%. For the first time, a systematic screen of convergently evolved motifs in 102.000 protein-protein interactions with structural information is derived. With respect to this dataset, all cases related to viral mimicry of human interface bindings are identified. Finally, a library of 740 motif descriptors for binding site recognition - encoded as Hidden Markov Models - is generated and cross-validated. Tests for the significance of motifs are provided. The usefulness of descriptors for protein-ligand binding sites is demonstrated for the case of &amp;quot;ATP-binding&amp;quot;, where a precision of 89% is achieved, thus outperforming comparable motifs from PROSITE. In particular, a novel descriptor for a P-loop variant has been used to identify ATP-binding sites in 60 protein sequences that have not been annotated before by existing motif databases

The Schistosoma mansoni cyclophilin A epitope 107-121 induces a protective immune response against schistosomiasis

Great efforts have been made to identify promising antigens and vaccine formulations against schistosomiasis. Among the previously described Schistosoma vaccine candidates, cyclophilins comprise an interesting antigen that could be used for vaccine formulations. Cyclophilin A is the target for the cyclosporine A, a drug with schistosomicide activity, and its orthologue from Schistosoma japonicum induces a protective immune response in mice. Although Schistosoma mansoni cyclophilin A also represents a promising target for anti-schistosome vaccines, its potential to induce protection has not been evaluated. In this study, we characterized the cyclophilin A (SmCyp), initially described as Smp17.7, analyzed its allergenic potential using in vitro functional assays, and evaluated its ability to induce protection in mice when administered as an antigen using different vaccine formulations and strategies. Results indicated that SmCyp could be successfully expressed by mammalian cells and bacteria. The recombinant protein did not promote IgE-reporter system activation in vitro, demonstrating its probable safety for use in vaccine formulations. T and B-cell epitopes were predicted in the SmCyp sequence, with two of them located within the active isomerase site. The most immunogenic antigen, SmCyp (107–121), was then used for immunization protocols. Immunization with the SmCyp gene or protein failed to reduce parasite burden but induced an immune response that modulated the granuloma area. In contrast, immunization with the synthetic peptide SmCyp (107–121) significantly reduced worm burden (48–50%) in comparison to control group, but did not regulate liver pathology. Moreover, the protection observed in mice immunized with the synthetic peptide was associated with the significant production of antibodies against the SmCyp (107–121) epitope. Therefore, in this study, we identified an epitope within the SmCyp sequence that induces a protective immune response against the parasite, thus representing a promising antigen that could be used for vaccine formulation against schistosomiasis

Stage-specific proteomes from onchocerca ochengi, sister species of the human river blindness parasite, uncover adaptations to a nodular lifestyle

Despite 40 years of control efforts, onchocerciasis (river blindness) remains one of the most important neglected tropical diseases, with 17 million people affected. The etiological agent, Onchocerca volvulus, is a filarial nematode with a complex lifecycle involving several distinct stages in the definitive host and blackfly vector. The challenges of obtaining sufficient material have prevented high-throughput studies and the development of novel strategies for disease control and diagnosis. Here, we utilize the closest relative of O. volvulus, the bovine parasite Onchocerca ochengi, to compare stage-specific proteomes and host-parasite interactions within the secretome. We identified a total of 4260 unique O. ochengi proteins from adult males and females, infective larvae, intrauterine microfilariae, and fluid from intradermal nodules. In addition, 135 proteins were detected from the obligate Wolbachia symbiont. Observed protein families that were enriched in all whole body extracts relative to the complete search database included immunoglobulin-domain proteins, whereas redox and detoxification enzymes and proteins involved in intracellular transport displayed stage-specific overrepresentation. Unexpectedly, the larval stages exhibited enrichment for several mitochondrial-related protein families, including members of peptidase family M16 and proteins which mediate mitochondrial fission and fusion. Quantification of proteins across the lifecycle using the Hi-3 approach supported these qualitative analyses. In nodule fluid, we identified 94 O. ochengi secreted proteins, including homologs of transforming growth factor-β and a second member of a novel 6-ShK toxin domain family, which was originally described from a model filarial nematode (Litomosoides sigmodontis). Strikingly, the 498 bovine proteins identified in nodule fluid were strongly dominated by antimicrobial proteins, especially cathelicidins. This first high-throughput analysis of an Onchocerca spp. proteome across the lifecycle highlights its profound complexity and emphasizes the extremely close relationship between O. ochengi and O. volvulus The insights presented here provide new candidates for vaccine development, drug targeting and diagnostic biomarkers

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

<p>Abstract</p> <p>Background</p> <p>Tardigrades represent an animal phylum with extraordinary resistance to environmental stress.</p> <p>Results</p> <p>To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (<it>Milnesium tardigradum</it>, <it>Hypsibius dujardini</it>, <it>Echiniscus testudo</it>, <it>Tulinus stephaniae</it>, <it>Richtersius coronifer</it>) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer <url>http://waterbear.bioapps.biozentrum.uni-wuerzburg.de</url>. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data.</p> <p>Conclusion</p> <p>Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.</p

Peptidyl-Prolyl Isomerases as Promising Targets for Natural Product-Inspired Therapeutics.

Peptidyl-prolyl isomerases (PPIases) are a ubiquitously expressed super family of proteins that catalyze the cis/trans isomerization of prolyl bonds. Proline conformation acts as a regulatory switch during folding, activation and/or degradation of “clients” that include proteins with roles in cancer and neurodegeneration. PPIase inhibitors, therefore, could be important therapeutics. However, the active site of PPIases is shallow and well conserved between members, challenging selective inhibitor design. Despite this, macrocyclic natural products, including FK506, rapamycin and cyclosporin, bind PPIases with nanomolar or better affinity. These natural products possess an unusual “bifunctional” binding mode and bind two separate proteins simultaneously, which is critical for their activity. They exhibit remarkable pharmacological properties, including oral bioavailability, and rapidly accumulate in cells with widespread tissue distribution. Inspired by this mechanism, I synthesized a library of bifunctional molecules that bind both FKBP12 and HIV protease. Like FK506, we envisioned a model where coincident, high-level expression of both targets - as in HIV-infected lymphocytes with high levels of FKBP12 and HIV protease - would drive cyto-selective sequestration. The library possessed varying affinities for each target, retained passive membrane permeability, and had cellular activity. Molecules highly potent for FKBP12 and HIV protease were selectively taken up into relevant cell populations. Treatment with FKBP12 inhibitors limited partitioning of the molecules, while FKBP12 overexpression enhanced it. This suggests that avidity effects drive selective accumulation of bifunctional molecules in cells expressing high levels of both protein partners. We next focused on Pin1, a unique PPIase that binds prolines directly following phosphoserine/threonine residues. The requirement for an electronegative group in the Pin1 active site renders many inhibitors inactive from poor permeability. We hypothesized that the excellent pharmacological properties of FK506 might make it a suitable scaffold for engineering Pin1 inhibitors. However, FK506 has little affinity for Pin1, because it lacks the key electronegative region essential for Pin1 binding. To overcome this, we designed a novel semisynthetic strategy to modify FK506 at a position to improve affinity for Pin1. Installing a sulfamic acid significantly improved the affinity for Pin1 (>100-fold) in vitro. This strategy is designed to yield high-affinity membrane-permeable inhibitors of Pin1.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135789/1/dunyak_1.pd