Protein flexibility facilitates quaternary structure assembly and evolution.

The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components.

Separation and identification of lipids in the photosynthetic cousins of Apicomplexa Chromera velia and Vitrella brassicaformis.

The alveolate algae Chromera velia and Vitrella brassicaformis (chromerids) are the closest known phototrophic relatives to apicomplexan parasites. Apicomplexans are responsible for fatal diseases of humans and animals and severe economic losses. Availability of the genome sequences of chromerids together with easy and rapid culturing of C. velia makes this alga a suitable model for investigating elementary biochemical principals potentially important for the apicomplexan pathogenicity. Such knowledge allows us to better understand processes during the evolutionary transition from a phototrophy to the parasitism in Apicomplexa. We explored lipidomes of both algae using high-performance liquid chromatography with mass spectrometry or gas chromatography with flame ionization detection. A single high-performance liquid chromatography with mass spectrometry analysis in both ionization modes was sufficient for the separation and semi-quantification of lipids in chromerid algae. We detected more than 250 analytes belonging to five structural lipid classes, two lipid classes of precursors and intermediates, and triacylglycerols as storage lipids. Identification of suggested structures was confirmed by high-resolution mass spectrometry with an Orbitrap mass analyzer. An outstandingly high accumulation of storage triacylglycerols was found in both species. All the investigated aspects make C. velia a prospective organism for further applications in biotechnology.

Apicomplexan profilins in vaccine development applied to bovine neosporosis.

Neospora caninum, an intracellular protozoan parasite from the phylum Apicomplexa, is the etiologic agent of neosporosis, a disease considered as a major cause of reproductive loss in cattle and neuromuscular disease in dogs. Bovine neosporosis has a great economic impact in both meat and dairy industries, related to abortion, premature culling and reduced milk yields. Although many efforts have been made to restrain bovine neosporosis, there are still no efficacious control methods. Many vaccine-development studies focused in the apicomplexan proteins involved in the adhesion and invasion of the host cell. Among these proteins, profilins have recently emerged as potential vaccine antigens or even adjuvant candidates for several diseases caused by apicomplexan parasites. Profilins bind Toll-like receptors 11 and 12 initiating MyD88 signaling, that triggers IL-12 and IFN-γ production, which may promote protection against infection. Here we summarized the state-of-the-art of novel vaccine development based on apicomplexan profilins applied as antigens or adjuvants, and delved into recent advances on N. caninum vaccines using profilin in the mouse model and in cattle.

The ecology and evolution of animal medication: genetically fixed response versus phenotypic plasticity.

Animal medication against parasites can occur either as a genetically fixed (constitutive) or phenotypically plastic (induced) behavior. Taking the tritrophic interaction between the monarch butterfly Danaus plexippus, its protozoan parasite Ophryocystis elektroscirrha, and its food plant Asclepias spp. as a test case, we develop a game-theory model to identify the epidemiological (parasite prevalence and virulence) and environmental (plant toxicity and abundance) conditions that predict the evolution of genetically fixed versus phenotypically plastic forms of medication. Our model shows that the relative benefits (the antiparasitic properties of medicinal food) and costs (side effects of medicine, the costs of searching for medicine, and the costs of plasticity itself) crucially determine whether medication is genetically fixed or phenotypically plastic. Our model suggests that animals evolve phenotypic plasticity when parasite risk (a combination of virulence and prevalence and thus a measure of the strength of parasite-mediated selection) is relatively low to moderately high and genetically fixed medication when parasite risk becomes very high. The latter occurs because at high parasite risk, the costs of plasticity are outweighed by the benefits of medication. Our model provides a simple and general framework to study the conditions that drive the evolution of alternative forms of animal medication.

Flexible synthesis and evaluation of diverse anti-Apicomplexa cyclic peptides.

A modular approach to synthesize anti-Apicomplexa parasite inhibitors was developed that takes advantage of a pluripotent cyclic tetrapeptide scaffold capable of adjusting appendage and skeletal diversities in only a few steps (one to three steps). The diversification processes make use of selective radical coupling reactions and involve a new example of a reductive carbon-nitrogen cleavage reaction with SmI2. The resulting bioactive cyclic peptides have revealed new insights into structural factors that govern selectivity between Apicomplexa parasites such as Toxoplasma and Plasmodium and human cells.

Holding back the microfilament--structural insights into actin and the actin-monomer-binding proteins of apicomplexan parasites.

Parasites from the phylum Apicomplexa are responsible for several major diseases of man, including malaria and toxoplasmosis. These highly motile protozoa use a conserved actomyosin-based mode of movement to power tissue traversal and host cell invasion. The mode termed as 'gliding motility' relies on the dynamic turnover of actin, whose polymerisation state is controlled by a markedly limited number of identifiable regulators when compared with other eukaryotic cells. Recent studies of apicomplexan actin regulator structure-in particular those of the core triad of monomer-binding proteins, actin-depolymerising factor/cofilin, cyclase-associated protein/Srv2, and profilin-have provided new insights into possible mechanisms of actin regulation in parasite cells, highlighting divergent structural features and functions to regulators from other cellular systems. Furthermore, the unusual nature of apicomplexan actin itself is increasingly coming into the spotlight. Here, we review recent advances in understanding of the structure and function of actin and its regulators in apicomplexan parasites. In particular we explore the paradox between there being an abundance of unpolymerised actin, its having a seemingly increased potential to form filaments relative to vertebrate actin, and the apparent lack of visible, stable filaments in parasite cells.

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

Members of the phylum Apicomplexa are responsible for a wide range of diseases in humans and animals. The absence of an effective vaccine or safe curing drugs and the continuous emergence of resistant parasites to available treatments impose a high demand on the identification of novel targets for intervention against the apicomplexans. Protein kinases are considered attractive potential therapeutic targets not only against cancers but also to combat infectious diseases. The scope and aim of this review is to report on the recent progress in dissecting the impact of protein phosphorylation in regulating motility and invasion.

Behavioural resistance against a protozoan parasite in the monarch butterfly.

1. As parasites can dramatically reduce the fitness of their hosts, there should be strong selection for hosts to evolve and maintain defence mechanisms against their parasites. One way in which hosts may protect themselves against parasitism is through altered behaviours, but such defences have been much less studied than other forms of parasite resistance. 2. We studied whether monarch butterflies (Danaus plexippus L.) use altered behaviours to protect themselves and their offspring against the protozoan parasite Ophryocystis elektroscirrha (McLaughlin & Myers (1970), Journal of Protozoology, 17, p. 300). In particular, we studied whether (i) monarch larvae can avoid contact with infectious parasite spores; (ii) infected larvae preferentially consume therapeutic food plants when given a choice or increase the intake of such plants in the absence of choice; and (iii) infected female butterflies preferentially lay their eggs on medicinal plants that make their offspring less sick. 3. We found that monarch larvae were unable to avoid infectious parasite spores. Larvae were also not able to preferentially feed on therapeutic food plants or increase the ingestion of such plants. However, infected female butterflies preferentially laid their eggs on food plants that reduce parasite growth in their offspring. 4. Our results suggest that animals may use altered behaviours as a protection against parasites and that such behaviours may be limited to a single stage in the host-parasite life cycle. Our results also suggest that animals may use altered behaviours to protect their offspring instead of themselves. Thus, our study indicates that an inclusive fitness approach should be adopted to study behavioural defences against parasites.

Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa.

BACKGROUND: The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. RESULTS: We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. CONCLUSIONS: We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and propose specific hypotheses for experimental investigation. The apicomplexan-specific kinase features reported in this study can be used in the design of selective kinase inhibitors.

Identification and phylogenetic analysis of RNA binding domain abundant in apicomplexans or RAP proteins.

The RNA binding domain abundant in apicomplexans (RAP) is a protein domain identified in a diverse group of proteins, called RAP proteins, many of which have been shown to be involved in RNA binding. To understand the expansion and potential function of the RAP proteins, we conducted a hidden Markov model based screen among the proteomes of 54 eukaryotes, 17 bacteria and 12 archaea. We demonstrated that the domain is present in closely and distantly related organisms with particular expansions in Alveolata and Chlorophyta, and are not unique to Apicomplexa as previously believed. All RAP proteins identified can be decomposed into two parts. In the N-terminal region, the presence of variable helical repeats seems to participate in the specific targeting of diverse RNAs, while the RAP domain is mostly identified in the C-terminal region and is highly conserved across the different phylogenetic groups studied. Several conserved residues defining the signature motif could be crucial to ensure the function(s) of the RAP proteins. Modelling of RAP domains in apicomplexan parasites confirmed an ⍺/ß structure of a restriction endonuclease-like fold. The phylogenetic trees generated from multiple alignment of RAP domains and full-length proteins from various distantly related eukaryotes indicated a complex evolutionary history of this family. We further discuss these results to assess the potential function of this protein family in apicomplexan parasites.

Microneme proteins in apicomplexans.

Microneme secretion supports several key cellular processes including gliding motility, active cell invasion and migration through cells, biological barriers, and tissues. The modular design of microneme proteins enables these molecules to assist each other in folding and passage through the quality control system, accurately target to the micronemes, oligimerizing with other parasite proteins, and engaging a variety of host receptors for migration and cell invasion. Structural and biochemical analyses of MIC domains is providing new perspectives on how adhesion is regulated and the potentially distinct roles MICs might play in long or short range interactions during parasite attachment and entry. New access to complete genome sequences and ongoing advances in genetic manipulation should provide fertile ground for refining current models and defining exciting new roles for MICs in apicomplexan biology.

GpMyoF, a WD40 repeat-containing myosin associated with the myonemes of Gregarina polymorpha.

This study presents the first characterization of a WD40 repeat-containing myosin identified in the apicomplexan parasite Gregarina polymorpha. This 222.7 kDa myosin, GpMyoF, contains a canonical myosin motor domain, a neck domain with 6 IQ motifs, a tail domain containing short regions of predicted coiled-coil structure, and, most notably, multiple WD40 repeats at the C-terminus. In other proteins such repeats assemble into a beta-propeller structure implicated in mediating protein-protein interactions. Confocal microscopy suggests that GpMyoF is localized to the annular myonemes that gird the parasite cortex. Extraction studies indicate that this myosin shows an unusually tight association with the cytoskeletal fraction and can be solubilized only by treatment with high pH (11.5) or the anionic detergent sarkosyl. This novel myosin and its homologs, which have been identified in several related genera, appear to be unique to the Apicomplexa and represent the only myosins known to contain the WD40 domain. The function of this myosin in G. polymorpha or any of the other apicomplexan parasites remains uncertain.

Lipid biology of Apicomplexa: perspectives for new drug targets, particularly for Toxoplasma gondii.

Development of effective therapies for intracellular eukaryotic pathogens is a serious challenge, given the protected location of these pathogens and the similarity of their biology to that of the host. Identifying cellular processes that are unique to the parasite is therefore a crucial step towards defining appropriate drug targets. In the case of the apicomplexan parasite Toxoplasma gondii, the need to find alternative treatments is imperative because of the poor tolerability and frequent side-effects associated with existing therapeutic strategies. The discovery that the parasite uses lipid synthetic pathways which are different from, or absent in, the mammalian host is now driving a renewed interest in T. gondii lipid biology. Recent achievements in this field are promising and suggest that the elucidation of lipid pathways will provide new opportunities for designing potent antiparasitic strategies.

Binding and interaction of dinitroanilines with apicomplexan and kinetoplastid alpha-tubulin.

Despite years of use as commercial herbicides, it is still unclear how dinitroanilines interact with tubulin, how they cause microtubule disassembly, and why they are selectively active against plant and protozoan tubulin. In this work, through a series of computational studies, a common binding site of oryzalin, trifluralin, and GB-II-5 on apicomplexan and kinetoplastid alpha-tubulin is proposed. Furthermore, to investigate how dinitroanilines affect tubulin dynamics, molecular dynamics simulations of Leishmania alpha-tubulin with and without a bound dinitroaniline are performed. The results obtained provide insight into the molecular mechanism by which these compounds interact with tubulin and function to prevent microtubule assembly. Finally, to aid in the design of effective parasitic microtubule inhibitors, several novel dinitroaniline analogues are evaluated. The location of the binding site and the relative binding affinities of the dinitroanilines all agree well with experimental data.

Organelle-specific cochaperonins in apicomplexan parasites.

Protein maturation in eukaryotic organelles requires the type I chaperonin system; this comprises chaperonin 60 (Cpn60) and its cochaperonin. We have re-examined and revised the sequence of the nuclear genes specifying organellar cochaperonins in Plasmodium falciparum (Pf). One gene encodes a typical cochaperonin (PfCpn10) whereas the other (encoding PfCpn20) specifies two Cpn10 domains arranged in tandem as in plant chloroplasts. Transfection experiments using fluorescent reporters showed specific localization of PfCpn10 to the mitochondrion and PfCpn20 to the plastid. As P. falciparum also has two Cpn60s, one of which is targeted specifically to the mitochondrion and the other exclusively to the plastid, each organelle has a distinct type I chaperonin system. Comparative sequence analysis extended these findings to several other apicomplexan parasites that have both a mitochondrion and a plastid. Phylogenetic analysis suggests the Cpn10s and Cpn20s of apicomplexans are independently monophyletic. The apicomplexan Cpn10 is phylogenetically related to other mitochondrial versions but a significant relationship between apicomplexan Cpn20s and other cochaperonins was not established.

Iron superoxide dismutases in eukaryotic pathogens: new insights from Apicomplexa and Trypanosoma structures.

Prior studies have highlighted the potential of superoxide dismutases as drug targets in eukaryotic pathogens. This report presents the structures of three iron-dependent superoxide dismutases (FeSODs) from Trypanosoma cruzi, Leishmania major and Babesia bovis. Comparison with existing structures from Plasmodium and other trypanosome isoforms shows a very conserved overall fold with subtle differences. In particular, structural data suggest that B. bovis FeSOD may display similar resistance to peroxynitrite-mediated inactivation via an intramolecular electron-transfer pathway as previously described in T. cruzi FeSOD isoform B, thus providing valuable information for structure-based drug design. Furthermore, lysine-acetylation results in T. cruzi indicate that acetylation occurs at a position close to that responsible for the regulation of acetylation-mediated activity in the human enzyme.

Towards a molecular understanding of the apicomplexan actin motor: on a road to novel targets for malaria remedies?

Apicomplexan parasites are the causative agents of notorious human and animal diseases that give rise to considerable human suffering and economic losses worldwide. The most prominent parasites of this phylum are the malaria-causing Plasmodium species, which are widespread in tropical and subtropical regions, and Toxoplasma gondii, which infects one third of the world's population. These parasites share a common form of gliding motility which relies on an actin-myosin motor. The components of this motor and the actin-regulatory proteins in Apicomplexa have unique features compared with all other eukaryotes. This, together with the crucial roles of these proteins, makes them attractive targets for structure-based drug design. In recent years, several structures of glideosome components, in particular of actins and actin regulators from apicomplexan parasites, have been determined, which will hopefully soon allow the creation of a complete molecular picture of the parasite actin-myosin motor and its regulatory machinery. Here, current knowledge of the function of this motor is reviewed from a structural perspective.

LCCL proteins of apicomplexan parasites.

The LCCL module is a conserved, autonomous protein-folding domain that has recently been found in several extracellular proteins of apicomplexan parasites including Plasmodium, Toxoplasma, Cryptosporidium and Theileria, identifying a new protein family in the Apicomplexa. The expression and structure of these modular proteins has fostered speculation about the roles of these novel molecules in immune evasion. Here, the current data and literature on the members of this protein family are reviewed, with a discussion on their possible roles in host-parasite interaction.

Mix and match modules: structure and function of microneme proteins in apicomplexan parasites.

Microneme organelles are found in the apical complex of all apicomplexan parasites and play an important role in the invasion process. The recent identification of microneme proteins from different apicomplexan genera has revealed a striking conservation of structural domains, some of which show functional complementation across species. This supports the idea that the mechanism of host cell invasion across the phylum is conserved not only morphologically, but also functionally at the molecular level. Here, we review and summarize these recent findings.

Suppression of chemiluminescence of eastern oyster (Crassostrea virginica) hemocytes by the protozoan parasite Perkinsus marinus.

Experiments were conducted to determine the ability of the protistan parasite, Perkinsus marinus, to inhibit chemiluminescence of hemocytes from the eastern oyster, Crassostrea virginica. Luminol-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen intermediates (ROI) generated by oyster hemocytes using zymosan as a stimulant. To determine whether P. marinus suppresses ROI evoked from zymosan-stimulated hemocytes, live or heat killed P. marinus in filtered estuarine water (YRW) (salinity = 20 ppt) were added to (1) zymosan-stimulated hemocytes after CL reached its peak, or (2) hemocytes at the same time as zymosan, and reduction of CL responses were recorded. In both tests, controls received only estuarine water. Live P. marinus meronts significantly suppressed ROI production by zymosan-stimulated hemocytes. The suppression of ROI production was dose dependent. Suppression of ROI production from zymosan-stimulated hemocytes by heat killed P. marinus was significantly less than by live P. marinus. Similarly, CL of hemocytes was reduced, though not significantly when hemocytes were exposed to YRW preincubated with P. marinus. When P. marinus meronts were used as a stimulant, no CL response was elicited. Results of this study suggest that P. marinus cells are able to suppress ROI release from oyster hemocytes, thus evading this component of the host's defense.