other Science:

Annotated Citations.

[note: “in Press”, “online advance publication”, etc., are indicated by “inPr_” preceeding journal name. “Final_” indicates a final version of an article previously downloaded in an “inPr_” format. “NA_” indicates journals for which our library system does not have an electronic subscription (as far as I can tell).]

1. Andreeva, A.V. and M.A. Kutuzov (2008), Protozoan protein tyrosine phosphatases. inPr_Int J Parasitol in Press. PMID: 18547579.

The aim of this review is to provide a synthesis of the published experimental data on protein tyrosine phosphatases from parasitic protozoa, in silico analysis based on the availability of completed genomes and to place available data for individual phosphatases from different unicellular parasites into the comparative and evolutionary context. We analysed the complement of protein tyrosine phosphatases (PTP) in several species of unicellular parasites that belong to Apicomplexa (Plasmodium; Cryptosporidium, Babesia, Theileria, and Toxoplasma), kinetoplastids (Leishmania and Trypanosoma spp.), as well as Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis and a microsporidium Encephalitozoon cuniculi. The analysis shows distinct distribution of the known families of tyrosine phosphatases in different species. Protozoan tyrosine phosphatases show considerable levels of divergence compared with their mammalian homologues, both in terms of sequence similarity between the catalytic domains and the structure of their flanking domains. This potentially makes them suitable targets for development of specific inhibitors with minimal effects on physiology of mammalian hosts.

2. Besteiro, S., D. Tonn, L. Tetley, G.H. Coombs, and J.C. Mottram (2008), The AP3 adaptor is involved in the transport of membrane proteins to acidocalcisomes of Leishmania. J Cell Sci 121(Pt 5): 561-70. PMID: 18252798.

Lysosomal function is crucial for the differentiation and infectivity of the parasitic protozoon Leishmania major. To study lysosomal biogenesis, an L. major mutant deficient in the delta subunit of the adaptor protein 3 (AP3 delta) complex was generated. Structure and proteolytic capacity of the lysosomal compartment were apparently unaffected in the AP3-deficient mutant; however, defects were identified in its acidocalcisomes. These are acidic organelles enriched in calcium and phosphorus, conserved from bacteria to eukaryotes, whose function remains enigmatic. The acidocalcisomes of the L. major mutant lacked membrane-bound proton pumps (notably V-H+-PPase), were less acidic than normal acidocalcisomes and devoid of polyphosphate, but contained a soluble pyrophosphatase. The mutant parasites were viable in vitro, but were unable to establish an infection in mice, which indicates a role for AP3 in determining–possibly through an acidocalcisome-related function–the virulence of the parasite. AP3 transport function has been linked previously to lysosome-related organelles such as platelet dense granules, which appear to share several features with acidocalcisomes. Our findings, implicating that AP3 has a role in transport to acidocalcisomes, thus provide further evidence that biogenesis of acidocalcisomes resembles that of lysosome-related organelles, and that both may have conserved origins.

3. De Silva, E.K., A.R. Gehrke, K. Olszewski, I. Leon, J.S. Chahal, M.L. Bulyk, and M. Llinas (2008), Specific DNA-binding by Apicomplexan AP2 transcription factors. Proc Natl Acad Sci U S A 105(24): 8393–8398. PMID: 18541913.

Malaria remains one of the most prevalent infectious diseases worldwide, affecting more than half a billion people annually. Despite many years of research, the mechanisms underlying transcriptional regulation in the malaria-causing Plasmodium spp., and in Apicomplexan parasites generally, remain poorly understood. In Plasmodium, few regulatory elements sufficient to drive gene expression have been characterized, and their cognate DNA-binding proteins remain unknown. This study characterizes the DNA-binding specificities of two members of the recently identified Apicomplexan AP2 (ApiAP2) family of putative transcriptional regulators from Plasmodium falciparum. The ApiAP2 proteins contain AP2 domains homologous to the well characterized plant AP2 family of transcriptional regulators, which play key roles in development and environmental stress response pathways. We assayed ApiAP2 protein-DNA interactions using protein-binding microarrays and combined these results with computational predictions of coexpressed target genes to couple these putative trans factors to corresponding cis-regulatory motifs in Plasmodium. Furthermore, we show that protein-DNA sequence specificity is conserved in orthologous proteins between phylogenetically distant Apicomplexan species. The identification of the DNA-binding specificities for ApiAP2 proteins lays the foundation for the exploration of their role as transcriptional regulators during all stages of parasite development. Because of their origin in the plant lineage, ApiAP2 proteins have no homologues in the human host and may prove to be ideal antimalarial targets.

4. Garcia, C.R., M.F. de Azevedo, G. Wunderlich, A. Budu, J.A. Young, and L. Bannister (2008), Plasmodium in the postgenomic era: new insights into the molecular cell biology of malaria parasites. NA_Int Rev Cell Mol Biol 266: 85-156. PMID: 18544493.

In this review, we bring together some of the approaches toward understanding the cellular and molecular biology of Plasmodium species and their interaction with their host red blood cells. Considerable impetus has come from the development of new methods of molecular genetics and bioinformatics, and it is important to evaluate the wealth of these novel data in the context of basic cell biology. We describe how these approaches are gaining valuable insights into the parasite-host cell interaction, including (1) the multistep process of red blood cell invasion by the merozoite; (2) the mechanisms by which the intracellular parasite feeds on the red blood cell and exports parasite proteins to modify its cytoadherent properties; (3) the modulation of the cell cycle by sensing the environmental tryptophan-related molecules; (4) the mechanism used to survive in a low Ca(2+) concentration inside red blood cells; (5) the activation of signal transduction machinery and the regulation of intracellular calcium; (6) transfection technology; and (7) transcriptional regulation and genome-wide mRNA studies in Plasmodium falciparum.

5. Issar, N., E. Roux, D. Mattei, and A. Scherf (2008), Identification of a novel post-translational modification in Plasmodium falciparum: Protein SUMOylation in different cellular compartments. inPr_Cell Microbiol in Press. PMID: 18547337.

SUMO (Small Ubiquitin-like MOdifier) conjugation is a post-translational modification implicated in a variety of cellular functions including transcriptional regulation, nuclear location and signal transduction. Sumoylation, although conserved and vital in eukaryotes, has not been studied in malaria parasites. Here we identify SUMO conjugation of blood stage parasites of P. falciparum. Antibodies raised against synthetic peptides of the plasmodial SUMO orthologue PfSUMO, a 100 amino acid protein, reacted with distinctive sub-cellular compartments of the parasitized erythrocyte during blood stage development. Anti-PfSUMO stains the nucleus and parasite cytoplasm. We also found antibody reactivity in the host cell cytoplasm with the parasite-derived structures called Maurer’s clefts. Anti-PfSUMO reacts in Western blot with a number of blood stage proteins ranging from approx. 40 to 250 kDa. Parasites expressing FLAG-tagged PfSUMO gave similar results in Immunofluorescence assay (IFA) and Western blots. In addition, we show that anti-PfSUMO identified PfSir2, a telomere-associated nuclear protein involved in var gene silencing, as a target for sumoylation. Furthermore, LC-MS/MS analysis of a two-step Immunoprecipitation with anti-FLAG and anti-PfSUMO antibodies reveals a number of putative P. falciparum sumoylated proteins. Our results imply that SUMO conjugation has an essential function in a number of different biological processes in P. falciparum.

6. Mackenzie, J.J., N.D. Gomez, S. Bhattacharjee, S. Mann, and K. Haldar (2008), A Plasmodium falciparum Host-Targeting Motif Functions in Export during Blood Stage Infection of the Rodent Malarial Parasite Plasmodium berghei. PLoS ONE 3(6): e2405. PMID: 18545649.

Plasmodium falciparum (P. falciparum) secretes hundreds of proteins-including major virulence proteins-into the host erythrocyte. In order to reach the host cytoplasm, most P. falciparum proteins contain an N terminal host-targeting (HT) motif composed of 11 amino acids. In silico analyses have suggested that the HT motif is conserved throughout the Plasmodium species but experimental evidence only exists for P. falciparum. Here, we show that in the rodent malaria parasite Plasmodium berghei (P. berghei) a reporter-like green fluorescent protein expressed by the parasite can be exported to the erythrocyte cytoplasm in a HT-specific manner. This provides the first experimental proof that the HT motif can function as a signal for protein delivery to the erythrocyte across Plasmodium species. Further, it suggests that P. berghei may serve as a model for validation of P. falciparum secretome proteins. We also show that tubovesicular membranes extend from the vacuolar parasite into the erythrocyte cytoplasm and speculate that these structures may facilitate protein export to the erythrocyte.

7. Ponts, N., J. Yang, Chung, D., W.Prudhomme, J. Girke, T. Horrocks, P. Le Roch, K. G. (2008), Deciphering the ubiquitin-mediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence. PLoS ONE (6): e2386. PMID: 18545708.

BACKGROUND: Reversible modification of proteins through the attachment of ubiquitin or ubiquitin-like modifiers is an essential post-translational regulatory mechanism in eukaryotes. The conjugation of ubiquitin or ubiquitin-like proteins has been demonstrated to play roles in growth, adaptation and homeostasis in all eukaryotes, with perturbation of ubiquitin-mediated systems associated with the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the use of an HMM search of functional Pfam domains found in the key components of the ubiquitin-mediated pathway necessary to activate and reversibly modify target proteins in eight apicomplexan parasitic protozoa for which complete or late-stage genome projects exist. In parallel, the same search was conducted on five model organisms, single-celled and metazoans, to generate data to validate both the search parameters employed and aid paralog classification in Apicomplexa. For each of the 13 species investigated, a set of proteins predicted to be involved in the ubiquitylation pathway has been identified and demonstrates increasing component members of the ubiquitylation pathway correlating with organism and genome complexity. Sequence homology and domain architecture analyses facilitated prediction of apicomplexan-specific protein function, particularly those involved in regulating cell division during these parasite’s complex life cycles. CONCLUSIONS/SIGNIFICANCE: This study provides a comprehensive analysis of proteins predicted to be involved in the apicomplexan ubiquitin-mediated pathway. Given the importance of such pathway in a wide variety of cellular processes, our data is a key step in elucidating the biological networks that, in part, direct the pathogenicity of these parasites resulting in a massive impact on global health. Moreover, apicomplexan-specific adaptations of the ubiquitylation pathway may represent new therapeutic targets for much needed drugs against apicomplexan parasites.

8. Sibley, L. D. and J. W. Ajioka (2008), Population Structure of Toxoplasma gondii: Clonal Expansion Driven by Infrequent Recombination and Selective Sweeps. inPr_Annu Rev Microbiol in Press. PMID: 18544039.

Toxoplasma gondii is among the most successful parasites. It is capable of infecting all warm-blooded animals and causing opportunistic disease in humans. T. gondii has a striking clonal population structure consisting of three predominant lineages in North America and Europe. Clonality is associated with the recent emergence of a monomorphic version of Chr1a, which drove a selective genetic sweep within the past 10,000 years. Strains from South America diverged from those in North America some 1-2 mya; recently, however, the monomorphic Chr1a has extended into regions of South America, where it is also associated with clonality. The recent spread of a few dominant lineages has dramatically shaped the population structure of T. gondii and has resulted in most lineages sharing a highly pathogenic nature. Understanding the factors that have shaped the population structure of T. gondii has implications for the emergence and transmission of human pathogens. Expected final online publication date for the Annual Review of Microbiology Volume 62 is September 08, 2008. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

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Parasitology: __[1-10]

other Science: __[11-20]

Annotated Citations.

[note: “in Press”, “online advance publication”, etc., are indicated by “inPr_” preceeding journal name. “Final_” indicates a final version of an article previously downloaded in an “inPr_” format. “NA_” indicates journals for which our library system does not have an electronic subscription (as far as I can tell).]

1. Barragan, A. and N. Hitziger (2008), Transepithelial migration by Toxoplasma. NA_Subcell Biochem 47: 198-207. PMID: 18512353.

A hallmark of T. gondii infections is passage of parasites across restrictive biological barriers–intestine, blood-brain barrier, blood-retina barrier and placenta-during primary infection or reactivation of chronic disease. Traversal of cellular barriers permits rapid dissemination of parasites to gain access to biologically restricted organs. This process involves active parasite motility and tightly regulated interactions between host cell receptors and parasite adhesins that facilitate paracellular transfer. Mounting evidence also suggests that parasites use migrating leukocytes as Trojan horses to disseminate in the organism while avoiding immune attack. Thus, the interaction of Toxoplasma with biological barriers is a determinant factor of human toxoplasmosis. The elucidation of determinants involved in the process of migration may reveal virulence factors and novel therapeutic targets to combat disease.

2. Binder, E.M., V. Lagal, and K. Kim (2008), The prodomain of Toxoplasma gondii GPI-anchored subtilase TgSUB1 mediates its targeting to micronemes. inPr_Traffic in Press. PMID: 18532988.

Subtilisin-like proteases have been proposed to play an important role for parasite survival in Toxoplasma gondii and Plasmodium falciparum. The T. gondii subtilase TgSUB1 is located in the microneme, an apical secretory organelle whose contents mediate adhesion to the host during invasion. TgSUB1 is predicted to contain a glycosyl-phosphatidylinositol anchor (GPI). This is unusual, as Toxoplasma GPI anchored proteins are targeted to the parasite’s surface. Here, we report that the subtilase TgSUB1 is indeed a GPI anchored protein, but contains dominant microneme targeting signals. Accurate targeting of TgSUB1 to the micronemes is dependent upon several factors including promoter strength and timing, accurate processing, and folding. We analyzed the targeting domains of TgSUB1 using TgSUB1 deletion constructs and chimeras made between TgSUB1 and reporter proteins. The TgSUB1 prodomain is responsible for trafficking to the micronemes and is sufficient for targeting a reporter protein to the micronemes. Trafficking is dependent upon correct folding or other context-dependent conformation, as the prodomain expressed alone is unable to reach the micromenes. Therefore, TgSUB1 is a novel example of a GPI anchored protein in T. gondii that bypasses the GPI dependent surface trafficking pathway to traffic to micronemes, specialized regulated secretory organelles.

3. Carruthers, V.B. and F.M. Tomley (2008), Microneme proteins in apicomplexans. NA_Subcell Biochem 47: 33-45. PMID: 18512339.

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.

4. Eggleston, T.L., E. Fitzpatrick, and K.M. Hager (2008), Parasitology as a teaching tool: isolation of apicomplexan cysts from store-bought meat. CBE Life Sci Educ 7(2): 184-92. PMID: 18519609.

Parasites are extremely prevalent not only in terms of historical medical and research references but also in terms of geographic distribution. Human parasites, such as Trichuris trichiura, Ascaris lumbricoides, and hookworms have remained relatively unchanged in their geographic distribution in the past 50 yr (Roberts and Schmidt, 2000). An outstanding example of this widespread distribution is the protozoan parasite Toxoplasma gondii. T. gondii infects humans worldwide and is broadly distributed in animals as varied as polar bears, bobcats, pigs, and even in marine mammals such as seals and otters (Fayer et al., 2004). In practical terms, this cosmopolitan distribution means that T. gondii is readily isolated from raw meat purchased at a local grocery store and thus a parasitology lesson is as close as your nearest checkout line. The primary focus of this essay is on the development of an assay to isolate apicomplexan cysts. We believe as an outcome of using this assay, teachers will be able to use the isolation of Toxoplasma and related apicomplexan tissue cysts as a means to teach food safety, and to introduce some basic concepts about infectious disease and cell biology.

5. Guler, J.L., E. Kriegova, T.K. Smith, J. Lukes, and P.T. Englund (2008), Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei. Mol Microbiol 67(5): 1125-42. PMID: 18221265.

Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome-mediated respiration. This reduction was explained by RNAi-mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function.

6. Mancio-Silva, L., A.P. Rojas-Meza, M. Vargas, A. Scherf, and R. Hernandez-Rivas (2008), Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum. J Cell Sci 121(Pt 12): 2046-53. PMID: 18525026.

Telomeres have the capacity to recruit proteins that facilitate the spreading of heterochromatin into subtelomeric DNA regions. In the human protozoan pathogen Plasmodium falciparum, the telomere-associated protein Sir2 has been shown to control the silencing of members of virulence genes at some, but not all, chromosome-end loci, indicating that additional proteins are involved in telomere position effect. Here, we identified, in P. falciparum, a novel telomere-associated protein that displays homology with the origin-of-recognition-complex 1 protein Orc1. Antibodies raised against this P. falciparum protein localized to telomeric clusters in the nuclear periphery and the nucleolus. It was found that, prior to DNA replication, P. falciparum Orc1 and Sir2 undergo drastic subcellular reorganization, such as dissociation from the telomere cluster and spreading into the nucleus and parasite cytoplasm. Relocation of Orc1 and Sir2 was also linked to the partial dissociation of telomere clusters. Super gel-shift and chromatin-immunoprecipitation experiments showed the physical association of Orc1 with telomere repeats but revealed a differential association with adjacent non-coding repeat DNA elements. Our data suggest that Plasmodium telomeres might fold back and that Orc1 cooperates with Sir2 in telomeric silencing.

7. Matuschewski, K. and H. Schuler (2008), Actin/myosin-based gliding motility in apicomplexan parasites. NA_Subcell Biochem 47: 110-20. PMID: 18512346.

Apicomplexan parasites move and actively enter host cells by substrate-dependent gliding motility, an unusual form of eukaryotic locomotion that differs fundamentally from the motility of prokaryotic and viral pathogens. Recent research has uncovered some of the cellular and molecular mechanisms underlying parasite motility, transmigration, and cell invasion during life cycle progression. The gliding motor machinery is embedded between the plasma membrane and the inner membrane complex, a unique double membrane layer. It consists ofimmobilized unconventional myosins, short actin stubs, and TRAP-family invasins. Assembly of this motor machinery enables force generation between parasite cytoskeletal components and an extracellular substratum. Unique properties of the individual components suggest that the rational design of motility inhibitors may lead to new intervention strategies to combat some of the most devastating human and livestock diseases.

8. McRobert, L., C.J. Taylor, W. Deng, Q.L. Fivelman, R.M. Cummings, S.D. Polley, O. Billker, and D.A. Baker (2008), Gametogenesis in Malaria Parasites Is Mediated by the cGMP-Dependent Protein Kinase. PLoS Biol 6(6): e139. PMID: 18532880.

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca(2+)) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.

9. Mital, J. and G.E. Ward (2008), Current and emerging approaches to studying invasion in apicomplexan parasites. NA_Subcell Biochem 47: 1-32. PMID: 18512338.

In this chapter, we outline the tools and techniques available to study the process of host cell invasion by apicomplexan parasites and we provide specific examples of how these methods have been used to further our understanding of apicomplexan invasive mechanisms. Throughout the chapter we focus our discussion on Toxoplasmagondii, because T. gondii is the most experimentally accessible model organism for studying apicomplexan invasion (discussed further in the section, “Toxoplasma as a Model Apicomplexan”) and more is known about invasion in T. gondii than in any other apicomplexan.

10. Sinai, A.P. (2008), Biogenesis of and activities at the Toxoplasma gondii parasitophorous vacuole membrane. NA_Subcell Biochem 47: 155-64. PMID: 18512349.

Apicomplexan parasites like Toxoplasma gondii are distinctive in their utilization of para site encoded motor systems to invade cells. Invasion results in the establishment of the parasitophorous vacuole (PV) within the infected cell. Most apicomplexans complete their intracellular tenure within the infected cell in the PV that is demarcated from the host cytoplasm by the parasitophorous vacuole membrane (PVM). In this chapter I focus on the events surrounding the formation of the PVM and selected activities attributed to it. Its central role as the interface between the parasite and its immediate environment, the host cytoplasm, is validated by the diversity of functions attributed to it. While functions in structural organization, nutrient acquisitions and signaling have been defined their molecular bases remain largely unknown. Several recent studies and the decoding of the Toxoplasma genome have set the stage for a rapid expansion in our understanding of the role of the PVM in parasite biology. Toxoplasma gondii, like all apicomplexan parasites are obligate intracellular pathogens. This family of parasites utilize their own actin-myosin based motor systems to gain entry into susceptible cells establishing themselves, in some cases transiently (e.g., Theileria spp) in specialized vacuolar compartment, the parasitophorous vacuole (PV). The T. gondii PV is highly dynamic compartment defining the replication permissive niche for the parasite. The delimiting membrane defining the parasitophorous vacuole, the parasitophorous vacuole membrane or PVM is increasingly being recognized as a specialized “organelle” that in the context of the infected cell is extracorporeal to the parent organism, the parasite. A systematic study of this enigmatic organelle has been severely limited by several issues. Primary among these is the fact that it is formed only in the context of the infected cell thereby limiting the amount of material. Secondly, unlike other cellular organelles that can often be purified by conventional approaches, the PVM, cannot be purified away from host cell organelles (see below). In spite of these significant obstacles considerable progress has been made in recent years toward understanding the biogenesis of the PVM, identification of its protein complement and the characterization of activities within it. These studies demonstrate that the PVM, on its own and by virtue of its interactions with cellular components, plays critical functions in the structural integrity of the vacuole, nutrient acquisition and the manipulation of cellular functions. In addition it appears that the repertoire of activities at the PVM is likely to be plastic reflecting temporal changes associated with the replicative phase of parasite growth. Finally, the PVM likely forms the foundation for the cyst wall as the parasite differentiates in the establishment of latent infection. As the critical border crossing between the parasite and invaded cell the study of the PVM provides a fertile area for new investigation aided by the recent decoding of the Toxoplasma genome (available at wwww.ToxoDB.org) and the application of proteomic analyses to basic questions in parasite biology.

11. Bereiter-Hahn, J., M. Voth, S. Mai, and M. Jendrach (2008), Structural implications of mitochondrial dynamics. inPr_Biotechnol J in Press. PMID: 18512869.

Mitochondrial components are continuously distributed throughout the whole chondriome of a cell by fusion and fission. Thus, a single mitochondrion represents a transient fraction of the chondriome. Mitochondrial dynamics are responsible for intracellular distribution and reaction of mitochondria to functional requirements. Dynamics occur on different levels: overall morphology, inner membrane-matrix compartment, turnover and rearrangements of mitochondrial proteins and DNA. Electron micrographs of serial sections of human umbilical vein endothelial cells reveal perinuclear mitochondria of extreme length and with branches in those cells that also have short peripheral mitochondria. Interactions of mitochondria with cytoskeletal elements are revealed in cells treated with cytochalasin D to destroy actin fibrillar structures or after disassembling microtubule by nocodazole. In the latter case mitochondria not only become immobilized, they also acquire a multiple ring structure. In F-actin-disturbed cells, motility (shape changes in particular) is increased and the mitochondria become elongated. Mechanisms of how F-actin might render mitochondria immobile may involve dynamin-related protein 1 (DRP1) or interaction with anion channels. This may be responsible for the lack of mitochondrial motility in senescent cells. Fusion between mitochondria revealed local fluctuations of mitochondrial red fluorescent protein (mtRFP), indicating novel fast inner membrane reorganizations. Mitochondrial dynamics result from a complex interplay between the molecular organization of the inner membrane-matrix complex and cytoskeletal elements outside.

12. Bose, M., B.P. Adams, R.M. Whittal, and H.S. Bose (2008), Identification of unknown protein complex members by radiolocalization and analysis of low-abundance complexes resolved using native polyacrylamide gel electrophoresis. Electrophoresis 29(4): 753-60. PMID: 18213604.

Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.

13. Bourbon, H.M. (2008), Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex. inPr_Nucleic Acids Res in Press. PMID: 18515835.

The multisubunit Mediator (MED) complex bridges DNA-bound transcriptional regulators to the RNA polymerase II (PolII) initiation machinery. In yeast, the 25 MED subunits are distributed within three core subcomplexes and a separable kinase module composed of Med12, Med13 and the Cdk8-CycC pair thought to control the reversible interaction between MED and PolII by phosphorylating repeated heptapeptides within the Rpb1 carboxyl-terminal domain (CTD). Here, MED conservation has been investigated across the eukaryotic kingdom. Saccharomyces cerevisiae Med2, Med3/Pgd1 and Med5/Nut1 subunits are apparent homologs of metazoan Med24/Trap100, Med27/Crsp34 and Med29/Intersex, respectively, and these and other 30 identified human MED subunits have detectable counterparts in the amoeba Dictyostelium discoideum, indicating that none is specific to metazoans. Indeed, animal/fungal subunits are also conserved in plants, green and red algae, entamoebids, oomycetes, diatoms, apicomplexans, ciliates and the ‘deep-branching’ protists Trichomonas vaginalis and Giardia lamblia. Surprisingly, although lacking CTD heptads, T. vaginalis displays 44 MED subunit homologs, including several CycC, Med12 and Med13 paralogs. Such observations have allowed the identification of a conserved 17-subunit framework around which peripheral subunits may be assembled, and support a very ancient eukaryotic origin for a large, four-module MED. The implications of this comprehensive work for MED structure-function relationships are discussed.

14. Meyer, E.H., N.L. Taylor, and A.H. Millar (2008), Resolving and identifying protein components of plant mitochondrial respiratory complexes using three dimensions of gel electrophoresis. J Proteome Res 7(2): 786-94. PMID: 18189341.

Analyzing highly hydrophobic proteins is a challenge for identification protocols based on gel separation and mass spectrometry. We combined Blue Native and 2D tricine gel electrophoresis to allow separation and identification of respiratory complex subunits from Arabidopsis mitochondria. We identified many of the highly hydrophobic mitochondrion-encoded subunits (GRAVY scores between +0.6 to +1.4) and also found a number of nucleus-encoded proteins associated with complex I for the first time in plants.

15. Nakamoto, R.K., J.A. Baylis Scanlon, and M.K. Al-Shawi (2008), The rotary mechanism of the ATP synthase. inPr_Arch Biochem Biophys in Press. PMID: 18515057.

The F(0)F(1) ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F(0) sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium.

16. Pisliakov, A.V., P.K. Sharma, Z.T. Chu, M. Haranczyk, and A. Warshel (2008), Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase. Proc Natl Acad Sci U S A 105(22): 7726-31. PMID: 18509049.

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is one of the challenges of modern biophysics. Despite promising mechanistic proposals, most works have not related the activation barriers of the different assumed steps to the protein structure, and there has not been a physically consistent model that reproduced the barriers needed to create a working pump. This work reevaluates the activation barriers for the primary proton transfer (PT) steps by calculations that reflect all relevant free energy contributions, including the electrostatic energies of the generated charges, the energies of water insertion, and large structural rearrangements of the donor and acceptor. The calculations have reproduced barriers that account for the directionality and sequence of events in the primary PT in CcO. It has also been found that the PT from Glu-286 (E) to the propionate of heme a(3) (Prd) provides a gate for an initial back leakage from the high pH side of the membrane. Interestingly, the rotation of E that brings it closer to Prd appears to provide a way for blocking competing pathways in the primary PT. Our study elucidates and quantifies the nature of the control of the directionality in the primary PT in CcO and provides instructive insight into the role of the water molecules in biological PT, showing that “bridges” of several water molecules in hydrophobic regions present a problem (rather than a solution) that is minimized in the primary PT.

17. Salinas, T., A.M. Duchene, and L. Marechal-Drouard (2008), Recent advances in tRNA mitochondrial import. inPr_Trends Biochem Sci in Press. PMID: 18513973.

In many eukaryotes, tRNA import from the cytosol into mitochondria is essential for mitochondrial biogenesis and, consequently, for cell viability. Recent work has begun to unravel the molecular mechanisms involved in tRNA transport in yeast, trypanosomatids and plants. The mechanisms of tRNA targeting to, and translocation through, the double mitochondrial membrane in addition to how selectivity and regulation of these processes are achieved are the main questions that have been addressed. The characterization of both direct and co-import mechanisms involving distinct protein-import factors is in agreement with a polyphyletic origin of tRNA import. Moreover, our increased understanding of the tRNA-import pathway has been exploited recently to rescue dysfunctions associated with mitochondrial tRNA mutations.

18. Sharma, V., M. Wikstrom, and L. Laakkonen (2008), Modeling the active-site structure of the cbb3-type oxidase from Rhodobacter sphaeroides. Biochemistry 47(14): 4221-7. PMID: 18338855.

The active site of the heme-copper oxidases comprises a redox-active high-spin heme and a tris-histidine copper center Cu B. Two amino acids in the close vicinity of the metals, a tyrosine and a tryptophan from helix 6, have been shown to be absolutely required for the catalytic function and should be considered part of the active site. Additionally, amino acid residues from interhelical loops strongly modify the activity. In a separate subfamily of heme-copper oxidases, the cbb 3-type oxidases, the metal centers are identical, the tyrosine is found in helix 7, but nothing is known of the corresponding tryptophan or of the involvement of the loop residues. We have observed a conserved aromatic cluster in the known oxidase structures, including the essential tryptophan and loop residues, and refined our earlier model of the cbb 3-type oxidase from Rhodobacter sphaeroides to test the feasibility of a similar structure. In the refined model, the interactions around the Delta-propionate of the high-spin heme resemble closely those seen in crystal structures of other terminal oxidases. Two alternative models (G- and C-models) that differ for the positioning of conserved tryptophans in helix 6, are presented. Molecular dynamics simulations on the catalytic subunit of the cbb 3-type oxidase model result in a conformational change of the active-site tyrosine, which may be related to different ligand-binding properties of the cbb 3-type oxidases. The relationship between sequence and functional data for defining the subfamily is discussed.

19. Sumimoto, H. (2008), Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. inPr_FEBS J in Press. PMID: 18513324.

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc(1) complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca(2+)-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin-NADP(+) reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1-4 subfamily in animals form a stable heterodimer with the membrane protein p22(phox), which functions as a docking site for the SH3 domain-containing regulatory proteins p47(phox), p67(phox), and p40(phox); the small GTPase Rac binds to p67(phox) (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67(phox)-like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation.

20. Wang, Y., M. Toei, and M. Forgac (2008), Analysis of the membrane topology of transmembrane segments in the C-terminal hydrophobic domain of the yeast vacuolar ATPase subunit a (Vph1p) by chemical modification. inPr_J Biol Chem in Press. PMID: 18508769.

The integral V0 domain of the vacuolar (H+)-ATPases (V-ATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100 kDa integral subunit of V0 that plays an essential role in proton translocation. In order to better define the membrane topology of subunit a, unique cysteine residues were introduced into a cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEG-mal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residue 602 and 620 and between residue 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734 and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C-terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7 and 8.

Parasitology: [1-12]

other Science: [13-22]

—–—

Annotated Citations.

[note: “in Press”, “online advance publication”, etc., are indicated by “inPr_” preceeding journal name. “Final_” indicates a final version of an article previously downloaded in an “inPr_” format. “NA_” indicates journals for which our library system does not have an electronic subscription (as far as I can tell).]

1. Blackman, M.J. (2008), Malarial proteases and host cell egress: an ‘emerging’ cascade. inPr_Cell Microbiol in Press. PMID: 18503638.

Malaria is a scourge of large swathes of the globe, stressing the need for a continuing effort to better understand the biology of its aetiological agent. Like all pathogens of the phylum Apicomplexa, the malaria parasite spends part of its life inside a host cell or cyst. It eventually needs to escape (egress) from this protective environment to progress through its life cycle. Egress of Plasmodium blood-stage merozoites, liver-stage merozoites and mosquito midgut sporozoites relies on protease activity, so the enzymes involved have potential as antimalarial drug targets. This review examines the role of parasite proteases in egress, in the light of current knowledge of the mechanics of the process. Proteases implicated in egress include the cytoskeleton degrading malarial proteases falcipain-2 and plasmepsin II, plus a family of putative papain-like proteases called SERA. Recent revelations have shown that activation of the SERA proteases may be triggered by regulated secretion of a subtilisin-like serine protease called SUB1. These findings are discussed in the context of the potential for development of new chemotherapeutics targeting this stage in the parasite’s life cycle.

2. Joubert, Y. and F. Joubert (2008), A structural annotation resource for the selection of putative target proteins in the malaria parasite. inPr_Malar J 7(in Press)(1): 90. PMID: 18500983.

ABSTRACT: BACKGROUND: Protein structure plays a pivotal role in elucidating mechanisms of parasite functioning and drug resistance. Moreover, protein structure aids the determination of protein function, which can together with the structure be used to identify novel drug targets in the parasite. However, various structural features in Plasmodium falciparum proteins complicate the experimental determination of protein structures. Limited similarity to proteins in the Protein Data Bank and the shortage of solved protein structures in the malaria parasite necessitate genome-scale structural annotation of P. falciparum proteins. Additionally, the annotation of a range of structural features facilitates the identification of suitable targets for experimental and computational studies. METHODS: An integrated structural annotation system was developed and applied to P. falciparum, Plasmodium vivax and Plasmodium yoelii. The annotation included searches for sequence similarity, patterns and domains in addition to the following predictions: secondary structure, transmembrane helices, protein disorder, low complexity, coiled-coils and small molecule interactions. Subsequently, candidate proteins for further structural studies were identified based on the annotated structural features. RESULTS: The annotation results are accessible through a web interface, enabling users to select groups of proteins which fulfil multiple criteria pertaining to structural and functional features. Analysis of features in the P. falciparum proteome showed that protein-interacting proteins contained a higher percentage of predicted disordered residues than non-interacting proteins. Proteins interacting with 10 or more proteins have a disordered content concentrated in the range of 60-100%, while the disorder distribution for proteins having only one interacting partner, was more evenly spread. CONCLUSIONS: A series of P. falciparum protein targets for experimental structure determination, comparative modelling and in silico docking studies were putatively identified. The system is available for public use, where researchers may identify proteins by querying with multiple physico-chemical, sequence similarity and interaction features.

3. Newby, Z.E., J. O’Connell Iii, Y. Robles-Colmenares, S. Khademi, L.J. Miercke, and R.M. Stroud (2008), Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum. inPr_Nat Struct Mol Biol in Press. PMID: 18500352.

The 2.05-A resolution structure of the aquaglyceroporin from the malarial parasite Plasmodium falciparum (PfAQP), a protein important in the parasite’s life cycle, has been solved. The structure provides key evidence for the basis of water versus glycerol selectivity in aquaporin family members. Unlike its closest homolog of known structure, GlpF, the channel conducts both glycerol and water at high rates, framing the question of what determines high water conductance in aquaporin channels. The universally conserved arginine in the selectivity filter is constrained by only two hydrogen bonds in GlpF, whereas there are three in all water-selective aquaporins and in PfAQP. The decreased cost of dehydrating the triply-satisfied arginine cation may provide the basis for high water conductance. The two Asn-Pro-Ala (NPA) regions of PfAQP, which bear rare substitutions to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), participate in preserving the orientation of the selectivity filter asparagines in the center of the channel.

4. Ostera, G., F. Tokumasu, F. Oliveira, J. Sa, T. Furuya, C. Teixeira, and J. Dvorak (2008), Plasmodium falciparum: Food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite. inPr_Exp Parasitol in Press. PMID: 18504040.

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.

5. Putz, S., P. Dolezal, G. Gelius-Dietrich, L. Bohacova, J. Tachezy, and K. Henze (2006), Fe-hydrogenase maturases in the hydrogenosomes of Trichomonas vaginalis. Eukaryot Cell 5(3): 579-86. PMID: 16524912.

Assembly of active Fe-hydrogenase in the chloroplasts of the green alga Chlamydomonas reinhardtii requires auxiliary maturases, the S-adenosylmethionine-dependent enzymes HydG and HydE and the GTPase HydF. Genes encoding homologous maturases had been found in the genomes of all eubacteria that contain Fe-hydrogenase genes but not yet in any other eukaryote. By means of proteomic analysis, we identified a homologue of HydG in the hydrogenosomes, mitochondrion-related organelles that produce hydrogen under anaerobiosis by the activity of Fe-hydrogenase, in the pathogenic protist Trichomonas vaginalis. Genes encoding two other components of the Hyd system, HydE and HydF, were found in the T. vaginalis genome database. Overexpression of HydG, HydE, and HydF in trichomonads showed that all three proteins are specifically targeted to the hydrogenosomes, the site of Fe-hydrogenase maturation. The results of Neighbor-Net analyses of sequence similarities are consistent with a common eubacterial ancestor of HydG, HydE, and HydF in T. vaginalis and C. reinhardtii, supporting a monophyletic origin of Fe-hydrogenase maturases in the two eukaryotes. Although Fe-hydrogenases exist in only a few eukaryotes, related Narf proteins with different cellular functions are widely distributed. Thus, we propose that the acquisition of Fe-hydrogenases, together with Hyd maturases, occurred once in eukaryotic evolution, followed by the appearance of Narf through gene duplication of the Fe-hydrogenase gene and subsequent loss of the Hyd proteins in eukaryotes in which Fe-hydrogenase function was lost.

6. Reece, S.E., D.R. Drew, and A. Gardner (2008), Sex ratio adjustment and kin discrimination in malaria parasites. Nature 453(7195): 609-14. PMID: 18509435.

Malaria parasites and related Apicomplexans are the causative agents of the some of the most serious infectious diseases of humans, companion animals, livestock and wildlife. These parasites must undergo sexual reproduction to transmit from vertebrate hosts to vectors, and their sex ratios are consistently female-biased. Sex allocation theory, a cornerstone of evolutionary biology, is remarkably successful at explaining female-biased sex ratios in multicellular taxa, but has proved controversial when applied to malaria parasites. Here we show that, as predicted by theory, sex ratio is an important fitness-determining trait and Plasmodium chabaudi parasites adjust their sex allocation in response to the presence of unrelated conspecifics. This suggests that P. chabaudi parasites use kin discrimination to evaluate the genetic diversity of their infections, and they adjust their behaviour in response to environmental cues. Malaria parasites provide a novel way to test evolutionary theory, and support the generality and power of a darwinian approach.

7. Saenz, F.E., B. Balu, J. Smith, S.R. Mendonca, and J.H. Adams (2008), The transmembrane isoform of Plasmodium falciparum MAEBL is essential for the invasion of Anopheles salivary glands. PLoS ONE 3(5): e2287. PMID: 18509478.

Malaria transmission depends on infective stages in the mosquito salivary glands. Plasmodium sporozoites that mature in midgut oocysts must traverse the hemocoel and invade the mosquito salivary glands in a process thought to be mediated by parasite ligands. MAEBL, a homologue of the transmembrane EBP ligands essential in merozoite invasion, is expressed abundantly in midgut sporozoites. Alternative splicing generates different MAEBL isoforms and so it is unclear what form is functionally essential. To identify the MAEBL isoform required for P. falciparum (NF54) sporozoite invasion of salivary glands, we created knockout and allelic replacements each carrying CDS of a single MAEBL isoform. Only the transmembrane form of MAEBL is essential and is the first P. falciparum ligand validated as essential for invasion of Anopheles salivary glands. MAEBL is the first P. falciparum ligand experimentally determined to be essential for this important step in the life cycle where the vector becomes infectious for transmitting sporozoites to people. With an increasing emphasis on advancing vector-based transgenic methods for suppression of malaria, it is important that this type of study, using modern molecular genetic tools, is done with the agent of the human disease. Understanding what P. falciparum sporozoite ligands are critical for mosquito transmission will help validate targets for vector-based transmission-blocking strategies.

8. Scholz, M. and M.J. Fraunholz (2008), A computational model of gene expression reveals early transcriptional events at the subtelomeric regions of the malaria parasite, Plasmodium falciparum. inPr_Genome Biol 9(in Press)(5): R88. PMID: 18505551.

ABSTRACT: BACKGROUND: The malaria parasite, Plasmodium falciparum, replicates asexually in a well-defined infection cycle within human erythrocytes (red blood cells, RBC). The intraerythrocytic developmental cycle (IDC) proceeds with a 48 hour periodicity. RESULTS: Based on available malaria microarray data, which monitored gene expression over one complete IDC in one-hour time intervals, we built a mathematical model of the IDC using a circular variant of non-linear principal component analysis. This model enables us to identify rates of expression change within the data and reveals early transcriptional events at the subtelomeres of the parasite’s nuclear chromosomes. CONCLUSIONS: A delay between subtelomeric and central gene activities suggests that key events of the IDC are initiated at the subtelomeric regions of the P. falciparum nuclear chromosomes.

9. Sharrock, W.W., R. Suwanarusk, U. Lek-Uthai, M.D. Edstein, V. Kosiavasee, T. Travers, A. Jaidee, K. Sriprawat, R.N. Price, F. Nosten, and B. Russell (2008), Plasmodium vivax trophozoites insensitive to chloroquine. inPr_Malar J 7(in Press)(1): 94. PMID: 18505560.

ABSTRACT: BACKGROUND: Plasmodium vivax is a major cause of malaria and is still primarily treated with chloroquine. Chloroquine inhibits the polymerization of haem to inert haemozoin. Free haem monomers are thought to catalyze oxidative damage to the Plasmodium spp. trophozoite, the stage when haemoglobin catabolism is maximal. However preliminary in vitro observations on P. vivax clinical isolates suggest that only ring stages (early trophozoites) are sensitive to chloroquine. In this study, the stage specific action of chloroquine was investigated in synchronous cryopreserved isolates of P. vivax. METHODS: The in vitro chloroquine sensitivity of paired ring and trophozoite stages from 11 cryopreserved P. vivax clinical isolates from Thailand and two Plasmodium falciparum clones (chloroquine resistant K1 and chloroquine sensitive FC27) was measured using a modified WHO microtest method and fluorometric SYBR Green I Assay. The time each stage was exposed to chloroquine treatment was controlled by washing the chloroquine off at 20 hours after the beginning of treatment. RESULTS: Plasmodium vivax isolates added to the assay at ring stage had significantly lower median IC50s to chloroquine than the same isolates added at trophozoite stage (median IC50 12 nM vs 415nM p<0.01). Although only 36% (4/11) of the SYBR Green I assays for P. vivax were successful, both microscopy and SYBR Green I assays indicated that only P. vivax trophozoites were able to develop to schizonts at chloroquine concentrations above 100nM. CONCLUSIONS: Data from this study confirms the diminished sensitivity of P. vivax trophozoites to chloroquine, the stage thought to be the target of this drug. These results raise important questions about the pharmacodynamic action of chloroquine, and highlight a fundamental difference in the activity of chloroquine between P. vivax and P. falciparum.

10. Sinnis, P. and F. Zavala (2008), The skin stage of malaria infection: biology and relevance to the malaria vaccine effort. Future Microbiol 3: 275-8. PMID: 18505393.

Plasmodium sporozoites, the infective stage of the malaria parasite, are injected into the mammalian host by mosquitoes and travel to the liver where they invade hepatocytes. Recent studies demonstrating that sporozoites are inoculated into the skin, remain there for hours before exiting and that 20% of the inoculum goes to the lymph node draining the inoculation site, suggest that there is a ‘skin stage’ to malaria infection that may set the stage for subsequent host responses to the parasite. Here, we present an overview of what is currently known about sporozoite-host interactions at the inoculation site and the draining lymph node, and discuss the impact of the skin stage of malaria on immunity to pre-erythrocytic stages and malaria vaccine design.

11. Tilley, L. and E. Hanssen (2008), A 3D view of the host cell compartment in P. falciparum-infected erythrocytes. inPr_Transfus Clin Biol in Press. PMID: 18501653.

The most deadly of the human malaria parasites, Plasmodium falciparum, invades the erythrocytes of its host and initiates a remarkable series of morphological rearrangements within the host cell cytoplasm. The mature erythrocyte is effectively a floating sack of haemoglobin with no endogenous protein synthesis or protein trafficking machinery. In order to colonise and remodel its extracellular space, the parasite generates a series of novel structures that are involved in the export of virulence factors to the surface of the host cell. These include extensions of the parasite’s vacuolar membrane, known as the tubulovesicular network, and structures referred to as Maurer’s clefts. Maurer’s clefts are convoluted collections of distorted discs that are tethered to the red blood cell membrane by structures with stalk-like profiles. Recently electron tomography has enabled visualisation – in three dimensions and at unprecedented resolution – the complexity of the membrane systems within the infected RBC cytoplasm.

12. Vincensini, L., G. Fall, L. Berry, T. Blisnick, and C. Braun Breton (2008), The RhopH complex is transferred to the host cell cytoplasm following red blood cell invasion by Plasmodium falciparum. inPr_Mol Biochem Parasitol in Press. PMID: 18508137.

The high-molecular mass rhoptry protein complex (PfRhopH), which comprises three distinct gene products, RhopH1, RhopH2, and RhopH3, is known to be secreted and transferred to the parasitophorous vacuole membrane upon invasion of a red blood cell by the malaria parasite Plasmodium falciparum. Here we show that the merozoite-acquired RhopH complex is also transferred to defined domains of the red blood cell cytoplasm, and possibly transiently associated with Maurer’s clefts. This is the first report of trafficking in the host cell cytoplasm for P. falciparum rhoptry proteins secreted upon red blood cell invasion. Based on its newly identified sub-cellular location and the phenotype of RhopH1 mutants, we propose that the RhopH complex participate in the assembly of the cytoadherence complex.

13. Brand, S.E., S. Rajagukguk, K. Ganesan, L. Geren, M. Fabian, D. Han, R.B. Gennis, B. Durham, and F. Millett (2007), A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase. Biochemistry 46(50): 14610-8. PMID: 18027981.

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine)2]2(quaterpyridine), (Ru2Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e., fully reducing the enzyme followed by reoxidation) just prior to photoreduction. Recent reports indicate transient changes in the redox behavior of the metal centers upon pulsing. The new photoreductant has a large quantum yield, allowing the kinetics data to be acquired in a single flash. The net charge of +4 on Ru2Z allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. The photoexcited state Ru(II*) of Ru2Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces CuA with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the “OH” state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru2Z, the oxidized OH state was formed within 5 ms. Ru2Z was then excited with a laser flash to inject one electron into CuA. Electron transfer from CuA –> heme a –> heme a3/CuB was monitored by optical spectroscopy, and the results were compared with the enzyme that had not been pulsed to the OH state. Pulsing had a significant effect in the case of the bovine oxidase, but this was not observed with the bacterial oxidases. Electron transfer from CuA to heme a occurred with a rate constant of 20,000 s-1 with the bovine cytochrome oxidase, regardless of whether the enzyme had been pulsed. However, electron transfer from heme a to the heme a3/CuB center in the pulsed form was 63% complete and occurred with biphasic kinetics with rate constants of 750 s-1 and 110 s-1 and relative amplitudes of 25% and 75%. In contrast, one-electron injection into the nonpulsed O form of the bovine oxidase was only 30% complete and occurred with monophasic kinetics with a rate constant of 90 s-1. This is the first indication of a difference between the fast form of the bovine oxidase and the pulsed OH form. No reduction of heme a3 is observed, indicating that CuB is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.

14. Bych, K., S. Kerscher, D.J. Netz, A.J. Pierik, K. Zwicker, M.A. Huynen, R. Lill, U. Brandt, and J. Balk (2008), The iron-sulphur protein Ind1 is required for effective complex I assembly. inPr_EMBO J in Press. PMID: 18497740.

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction ( approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.

15. Crofts, A.R., J.T. Holland, D. Victoria, D.R. Kolling, S.A. Dikanov, R. Gilbreth, S. Lhee, R. Kuras, and M.G. Kuras (2008), The Q-cycle reviewed: How well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex? inPr_Biochim Biophys Acta in Press. PMID: 18501698.

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.

16. Ding, M.G., C.A. Butler, S.A. Saracco, T.D. Fox, F. Godard, J.P. di Rago, and B.L. Trumpower (2008), Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins. inPr_Biochim Biophys Acta in Press. PMID: 18498758.

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.

17. Godman, J. and J. Balk (2008), Genome Analysis of Chlamydomonas reinhardtii Reveals The Existence of Multiple, Compartmentalized Iron-Sulfur Protein Assembly Machineries of Different Evolutionary Origins. Genetics 179(1): 59-68. PMID: 18493040.

The unicellular green alga Chlamydomonas reinhardtii is used extensively as a model to study eukaryotic photosynthesis, flagellar functions, and more recently the production of hydrogen as biofuel. Two of these processes, photosynthesis and hydrogen production, are highly dependent on iron-sulfur (Fe-S) enzymes. To understand how Fe-S proteins are assembled in Chlamydomonas, we have analyzed its recently sequenced genome for orthologs of genes involved in Fe-S cluster assembly. We found a total of 32 open reading frames, most single copies, that are thought to constitute a mitochondrial assembly pathway, mitochondrial export machinery, a cytosolic assembly pathway, and components for Fe-S cluster assembly in the chloroplast. The chloroplast proteins are also expected to play a role in the assembly of the H-cluster in [FeFe]-hydrogenases, together with the recently identified HydEF and HydG proteins. Comparison with the higher plant model Arabidopsis indicated a strong degree of conservation of Fe-S cofactor assembly pathways in the green lineage, the pathways being derived from different origins during the evolution of the photosynthetic eukaryote. As a haploid, unicellular organism with available forward and reverse genetic tools, Chlamydomonas provides an excellent model system to study Fe-S cluster assembly and its regulation in photosynthetic eukaryotes.

18. Goyon, V., R. Fronzes, B. Salin, J.P. di-Rago, J. Velours, and D. Brethes (2008), Yeast cells depleted in Atp14p fail to assemble Atp6p within the ATP synthase and exhibit altered mitochondrial cristae morphology. J Biol Chem 283(15): 9749-58. PMID: 18252710.

Within the yeast mitochondrial ATP synthase, subunit h is a small nuclear encoded protein belonging to the so-called “peripheral stalk” that connects the enzyme catalytic F(1) component to the mitochondrial inner membrane. This study examines the role of subunit h in ATP synthase function and assembly using a regulatable, doxycycline-repressible subunit h gene to overcome the strong instability of the mtDNA previously observed in strains lacking the native subunit h gene. Yeast cells expressing less than 3% of subunit h, but still containing intact mitochondrial genomes, grew poorly on respiratory substrates because of a major impairment of ATP synthesis originating from the ATP synthase, whereas the respiratory chain complexes were not affected. The lack of ATP synthesis in the subunit h-depleted (deltah) mitochondria was attributed to defects in the assembly/stability of the ATP synthase. A main feature of deltah-mitochondria was a very low content (<6%) in the mitochondrially encoded Atp6p subunit, an essential component of the enzyme proton channel, which was in large part because of a slowing down in translation. Interestingly, depletion of subunit h resulted in dramatic changes in mitochondrial cristae morphology, which further supports the existence of a link between the ATP synthase and the folding/biogenesis of the inner mitochondrial membrane.

19. Hoye, A.T., J.E. Davoren, P. Wipf, M.P. Fink, and V.E. Kagan (2008), Targeting mitochondria. Acc Chem Res 41(1): 87-97. PMID: 18193822.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely linked to degenerative diseases such as Alzheimer’s disease, Parkinson’s, neuronal death including ischemic and hemorrhagic stroke, acute and chronic degenerative cardiac myocyte death, and cancer. As a byproduct of oxidative phosphorylation, a steady stream of reactive species emerge from our cellular energy plants, the mitochondria. ROS and RNS potentially cause damage to all cellular components. Structure alteration, biomolecule fragmentation, and oxidation of side chains are trade-offs of cellular energy production. ROS and RNS escape results in the activation of cytosolic stress pathways, DNA damage, and the upregulation of JNK, p38, and p53. Incomplete scavenging of ROS and RNS particularly affects the mitochondrial lipid cardiolipin (CL), triggers the release of mitochondrial cytochrome c, and activates the intrinsic death pathway. Due to the active redox environment and the excess of NADH and ATP at the inner mitochondrial membrane, a broad range of agents including electron acceptors, electron donors, and hydride acceptors can be used to influence the biochemical pathways. The key to therapeutic value is to enrich selective redox modulators at the target sites. Our approach is based on conjugating nitroxides to segments of natural products with relatively high affinity for mitochondrial membranes. For example, a modified gramicidin S segment was successfully used for this purpose and proven to be effective in preventing superoxide production in cells and CL oxidation in mitochondria and in protecting cells against a range of pro-apoptotic triggers such as actinomycin D, radiation, and staurosporine. More importantly, these mitochondria-targeted nitroxide/gramicidin conjugates were able to protect against apoptosis in vivo by preventing CL oxidation induced by intestinal hemorrhagic shock. Optimization of nitroxide carriers could lead to a new generation of effective antiapoptotic agents acting at an early mitochondrial stage. Alternative chemistry-based approaches to targeting mitochondria include the use of proteins and peptides, as well as the attachment of payloads to lipophilic cationic compounds, sulfonylureas, anthracyclines, and other agents with proven or hypothetical affinities for mitochondria. Manganese superoxide dismutase (MnSOD), SS tetrapeptides with 2′,6′-dimethyltyrosine (Dmt) residues, rhodamine, triphenylphosphonium salts, nonopioid analgesics, adriamycin, and diverse electron-rich aromatics and stilbenes were used to influence mitochondrial biochemistry and the biology of aging. Some general structural principles for effective therapeutic agents are now emerging. Among these are the presence of basic or positively charged functional groups, hydrophobic substructures, and, most promising for future selective strategies, classes of compounds that are actively shuttled into mitochondria, bind to mitochondria-specific proteins, or show preferential affinity to mitochondria-specific lipids.

20. Kalanon, M. and G.I. McFadden (2008), The Chloroplast Protein Translocation Complexes of Chlamydomonas reinhardtii: A Bioinformatic Comparison of Toc and Tic Components in Plants, Green Algae and Red Algae. Genetics 179(1): 95-112. PMID: 18493043.

The recently completed genome of Chlamydomonas reinhardtii was surveyed for components of the chloroplast protein translocation complexes. Putative components were identified using reciprocal BlastP searches with the protein sequences of Arabidopsis thaliana as queries. As a comparison, we also surveyed the new genomes of the bryophyte Physcomitrella patens, two prasinophyte green algae (Ostreococcus lucimarinus and Ostreococcus tauri), the red alga Cyanidioschizon merolae, and several cyanobacteria. Overall, we found that the components of the import pathway are remarkably well conserved, particularly among the Viridiplantae lineages. Specifically, C. reinhardtii contained almost all the components found in A. thaliana, with two exceptions. Missing from C. reinhardtii are the C-terminal ferredoxin-NADPH-reductase (FNR) binding domain of Tic62 and a full-length, TPR-bearing Toc64. Further, the N-terminal domain of C. reinhardtii Toc34 is highly acidic, whereas the analogous region in C. reinhardtii Toc159 is not. This reversal of the vascular plant model may explain the similarity of C. reinhardtii chloroplast transit peptides to mitochondrial-targeting peptides. Other findings from our genome survey include the absence of Tic22 in both Ostreococcus genomes; the presence of only one Toc75 homolog in C. merolae; and, finally, a distinctive propensity for gene duplication in P. patens.

21. Klingenberg, M. (2008), The ADP and ATP transport in mitochondria and its carrier. inPr_Biochim Biophys Acta in Press. PMID: 18510943.

Different from some more specialised short reviews, here a general although not encyclopaedic survey of the function, metabolic role, structure and mechanism of the ADP/ATP transport in mitochondria is presented. The obvious need for an “old fashioned” review comes from the gateway role in metabolism of the ATP transfer to the cytosol from mitochondria. Amidst the labours, 40 or more years ago, of unravelling the role of mitochondrial compartments and of the two membranes, the sequence of steps of how ATP arrives in the cytosol became a major issue. When the dust settled, a picture emerged where ATP is exported across the inner membrane in a 1:1 exchange against ADP and where the selection of ATP versus ADP is controlled by the high membrane potential at the inner membrane, thus uplifting the free energy of ATP in the cytosol over the mitochondrial matrix. Thus the disparate energy and redox states of the two major compartments are bridged by two membrane potential responsive carriers to enable their symbiosis in the eukaryotic cell. The advance to the molecular level by studying the binding of nucleotides and inhibitors was facilitated by the high level of carrier (AAC) binding sites in the mitochondrial membrane. A striking flexibility of nucleotide binding uncovered the reorientation of carrier sites between outer and inner face, assisted by the side specific high affinity inhibitors. The evidence of a single carrier site versus separate sites for substrate and inhibitors was expounded. In an ideal setting principles of transport catalysis were elucidated. The isolation of intact AAC as a first for any transporter enabled the reconstitution of transport for unravelling, independently of mitochondrial complications, the factors controlling the ADP/ATP exchange. Electrical currents measured with the reconstituted AAC demonstrated electrogenic translocation and charge shift of reorienting carrier sites. Aberrant or vital para-functions of AAC in basal uncoupling and in the mitochondrial pore transition were demonstrated in mitochondria and by patch clamp with reconstituted AAC. The first amino acid sequence of AAC and of any eukaryotic carrier furnished a 6-transmembrane helix folding model, and was the basis for mapping the structure by access studies with various probes, and for demonstrating the strong conformation changes demanded by the reorientation mechanism. Mutations served to elucidate the function of residues, including the particular sensitivity of ATP versus ADP transport to deletion of critical positive charge in AAC. After resisting for decades, at last the atomic crystal structure of the stabilised CAT-AAC complex emerged supporting the principle predicted fold of the AAC but showing unexpected features relevant to mechanism. Being a snapshot of an extreme abortive “c-state” the actual mechanism still remains a conjecture.

22. Walther, D.M. and D. Rapaport (2008), Biogenesis of mitochondrial outer membrane proteins. inPr_Biochim Biophys Acta in Press. PMID: 18501716.

Mitochondria are surrounded by two distinct membranes: the outer and the inner membrane. The mitochondrial outer membrane mediates numerous interactions between the mitochondrial metabolic and genetic systems and the rest of the eukaryotic cell. Proteins of this membrane are nuclear-encoded and synthesized as precursor proteins in the cytosol. They are targeted to the mitochondria and inserted into their target membrane via various pathways. This review summarizes our current knowledge of the sorting signals for this specific targeting and describes the mechanisms by which the mitochondrial import machineries recognize precursor proteins, mediate their membrane integration and facilitate assembly into functional complexes.

Other Science: [16-19]

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Annotated Citations.

[note: “in Press”, “online advance publication”, etc indicated by “inPr_” preceeding journal name. “Final_” indicates a final version of an article previously downloaded in an “inPr_” format. “NA_” indicates journals for which our Library system does not have an electronic subscription (as far as I can tell).]

1. Akinyi, S., J. Gaona, E.V. Meyer, J.W. Barnwell, M.R. Galinski, and V. Corredor (2008), Phylogenetic and structural information on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in Plasmodium provides functional insights. Infect Genet Evol 8(2): 205-12. PMID: 18472435.

Plasmodium is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PDH) plays an important role in glycolysis and is, therefore, a potential target for antimalarial drug development. The g3pdh gene of nine Plasmodium species was sequenced from genomic DNA and the type and origin determined by phylogenetic analysis. Substitutions were analyzed over a wide phylogenetic spectrum in relation to the known three-dimensional structures of the P. falciparum and human proteins. Substitutions were found within the functional domains (Rossman NAD+-binding and catalytic domains). A number of replacements within the adenosyl-binding surfaces were found to be conserved within the Chromoalveolates, others in the Apicomplexa, and still others within the genus Plasmodium, all of which were different from the human sequence. These sites may prove to be of functional importance and provide insights for drug-targeting studies, as have other regions examined in Leishmania and Toxoplasma G3PDH research.

2. Aly, A.S., S.A. Mikolajczak, H.S. Rivera, N. Camargo, V. Jacobs-Lorena, M. Labaied, I. Coppens, and S.H. Kappe (2008), Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection. inPr_Mol Microbiol in Press. PMID: 18466298.

Malaria parasite sporozoites prepare for transmission to a mammalian host by upregulation of UIS (Upregulated in Infectious Sporozoites) genes. A number of UIS gene products are essential for the establishment of the intrahepatocytic niche. However, the factors that regulate the expression of genes involved in gain of infectivity for the liver are unknown. Herein, we show that a conserved Plasmodium sporozoite low complexity asparagine-rich protein, SAP1 (Sporozoite Asparagine-rich Protein1), has an essential role in malaria parasite liver infection. Targeted deletion of SAP1 in the rodent malaria parasite P. yoelii generated mutant parasites that traverse and invade hepatocytes normally but cannot initiate liver stage development in vitro and in vivo. Moreover, immunizations with Pysap1(-) sporozoites confer long lasting sterile protection against wildtype sporozoite infection. Strikingly, lack of SAP1 abolished expression of essential UIS genes including UIS3, UIS4 and P52 but not the constitutively expressed genes encoding, among others, sporozoite proteins CSP and TRAP. SAP1 localization to the cell interior but not the nucleus of sporozoites suggests its involvement in a posttranscriptional mechanism of gene expression control. These findings demonstrate that SAP1 is essential for liver infection possibly by functioning as a selective regulator controlling the expression of infectivity-associated parasite effector genes.

3. Black, C.G., N.I. Proellocks, L.M. Kats, B.M. Cooke, N. Mohandas, and R.L. Coppel (2008), In vivo studies support the role of trafficking and cytoskeletal-binding motifs in the interaction of MESA with the membrane skeleton of Plasmodium falciparum-infected red blood cells. inPr_Mol Biochem Parasitol in Press. PMID: 18482775.

In red blood cells (RBCs) infected with the malaria parasite Plasmodium falciparum, a 19-residue region of the mature parasite-infected erythrocyte surface antigen (MESA) associates with RBC cytoskeleton protein 4.1R; an interaction essential for parasite survival. This region in MESA is adjacent to a host targeting motif found in other malaria parasite proteins exported to the membrane skeleton. To demonstrate function of these motifs in vivo, regions of MESA fused to a reporter were expressed in malaria parasites. Immunochemical analyses confirmed the requirement for both motifs in the trafficking and interaction of MESA with the cytoskeleton and demonstrates their function in vivo.

4. Blythe, J.E., X.Y. Yam, C. Kuss, Z. Bozdech, A.A. Holder, K. Marsh, J. Langhorne, and P.R. Preiser (2008), Plasmodium falciparum STEVOR proteins are highly expressed in patient isolates and locate to the surface membrane of infected red blood cells and the apical tip of merozoites. inPr_Infect Immun in Press. PMID: 18474651.

The human parasite Plasmodium falciparum has the potential to express a vast repertoire of variant proteins on the surface of the infected red blood cell (iRBC). Variation in the expression pattern of these proteins is linked to antigenic variation and thereby evasion of host antibody-mediated immunity. The stevor multigene family codes for small variant antigens that are expressed in blood stage parasites where they can be detected in membranous structures called Maurer’s clefts (MC). Some studies have indicated that STEVOR may also be trafficked to the iRBC membrane. To address the location of STEVOR in more detail we have analyzed expression in several cultured parasite lines as well as in parasites obtained directly from patients. We detected STEVOR expression in a higher proportion of parasites recently isolated from patients than in cultured parasite lines and show that STEVOR is trafficked in schizont-stage parasites from the MC to the red cell cytosol and the iRBC membrane. Furthermore, STEVOR is also detected at the apical end of merozoites. Importantly we show that culture adapted parasites do not require STEVOR for survival. These findings provide new insights into the role of the STEVOR multigene family during both the schizont and merozoite stages of the parasite and highlight the importance of studying freshly isolated parasites, rather than parasite lines maintained in culture, when investigating potential mediators of host parasite interactions.

5. Githui, E.K., E.P. De Villiers, and A.G. McArthur (2008), Plasmodium possesses dynein light chain classes that are unique and conserved across species. inPr_Infect Genet Evol in Press. PMID: 18467191.

Plasmodium belongs to the phylum Apicomplexa. Within the Apicomplexa, Plasmodium, Toxoplasma and Cryptosporidium are parasites of considerable medical importance while Theileria and Eimeria are animal pathogens. P. falciparum is particularly important as it causes malaria, resulting in more than 1 million deaths each year. The malaria parasite actively invades the host cell in which it propagates and several proteins associated with the apical organelles have been implicated to be crucial in the invasion process. The biogenesis of the apical organelles is not well understood, but several studies indicate that microtubule-based vesicular transport is involved. Vesicular transport proteins are also present in Plasmodium and are presumed to be involved in transcellular transport in infected erythrocytes. Dynein is a multi-subunit motor protein involved in microtubule-based vesicular transport. In this study, we analyzed the cytoplasmic dynein light chains (Dlcs) of P. falciparum since they provide adaptor surface to the cargoes and are likely to be involved in differential transport. Dlcs consist of three different families: TcTex1/2, LC8 and LC7/roadblock. The data presented demonstrate that P. falciparum Dlcs sequences and functional domains show high sequence similarity within the species, but that only the Dlc group 1 ‘(LC8)’ has a high similarity to human orthologues. TcTex1 and LC7/roadblock have low similarity to human orthologues. This sequence variation could be targeted for vaccine or drug development.

6. Goldberg, A.V., S. Molik, A.D. Tsaousis, K. Neumann, G. Kuhnke, F. Delbac, C.P. Vivares, R.P. Hirt, R. Lill, and T.M. Embley (2008), Localization and functionality of microsporidian iron-sulphur cluster assembly proteins. Nature 452(7187): 624-8. PMID: 18311129.

Microsporidia are highly specialized obligate intracellular parasites of other eukaryotes (including humans) that show extreme reduction at the molecular, cellular and biochemical level. Although microsporidia have long been considered as early branching eukaryotes that lack mitochondria, they have recently been shown to contain a tiny mitochondrial remnant called a mitosome. The function of the mitosome is unknown, because microsporidians lack the genes for canonical mitochondrial functions, such as aerobic respiration and haem biosynthesis. However, microsporidial genomes encode several components of the mitochondrial iron-sulphur (Fe-S) cluster assembly machinery. Here we provide experimental insights into the metabolic function and localization of these proteins. We cloned, functionally characterized and localized homologues of several central mitochondrial Fe-S cluster assembly components for the microsporidians Encephalitozoon cuniculi and Trachipleistophora hominis. Several microsporidial proteins can functionally replace their yeast counterparts in Fe-S protein biogenesis. In E. cuniculi, the iron (frataxin) and sulphur (cysteine desulphurase, Nfs1) donors and the scaffold protein (Isu1) co-localize with mitochondrial Hsp70 to the mitosome, consistent with it being the functional site for Fe-S cluster biosynthesis. In T. hominis, mitochondrial Hsp70 and the essential sulphur donor (Nfs1) are still in the mitosome, but surprisingly the main pools of Isu1 and frataxin are cytosolic, creating a conundrum of how these key components of Fe-S cluster biosynthesis coordinate their function. Together, our studies identify the essential biosynthetic process of Fe-S protein assembly as a key function of microsporidian mitosomes.

7. Kandeel, M. and Y. Kitade (2008), Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase. inPr_J Biochem in Press PMID: 18477629.

The gene encoding for thymidylate kinase from Plasmodium falciparum was obtained by PCR and expressed in Escherichia coli and the enzyme was investigated as a possible new drug target. The enzyme is a homodimer exhibiting maximal kinase activity over a wide pH range of 7-9 and is characterized by marked stability. Compared with the human enzyme, the recombinant Plasmodium falciparum TMP kinase showed a broader spectrum of substrate specificity. The enzyme not only phosphorylates dTMP and dUMP but can also tolerate the bulkier purines dGMP, GMP and dIMP. Initial velocity studies showed that the K(m) values for TMP and dGMP are 22 and 30 muM, respectively. The turnover number k(cat(TMP)) was found to be 3.4 s(-1), a value indicating the higher catalytic efficiency of the plasmodium enzyme. From the present study we suggest that the design of appropriate inhibitors especially purine based compounds could have a selective inhibitory effect on the parasite enzyme.

8. Kotaka, M., H. Ye, R. Alag, G. Hu, Z. Bozdech, P.R. Preiser, H.S. Yoon, and J. Lescar (2008), Crystal Structure of the FK506 Binding Domain of Plasmodium falciparum FKBP35 in Complex with FK506. inPr_Biochemistry in Press. PMID: 18465874.

The emergence of multi-drug-resistant strains of Plasmodium parasites has prompted the search for alternative therapeutic strategies for combating malaria. One possible strategy is to exploit existing drugs as lead compounds. FK506 is currently used in the clinic for preventing transplant rejection. It binds to a alpha/beta protein module of approximately 120 amino acids known as the FK506 binding domain (FKBD), which is found in various organisms, including human, yeast, and Plasmodium falciparum (PfFKBD). Antiparasitic effects of FK506 and its analogues devoid of immunosuppressive activities have been demonstrated. We report here the crystallographic structure at 2.35 A resolution of PfFKBD complexed with FK506. Compared to the human FKBP12-FK506 complex reported earlier, the structure reveals structural differences in the beta5-beta6 segment that lines the FK506 binding site. The presence in PfFKBD of Cys-106 and Ser-109 (substituting for His-87 and Ile-90, respectively, in human FKBP12), which are 4-5 A from the nearest atom of the FK506 compound, suggests possible routes for the rational design of analogues of FK506 with specific antiparasitic activity. Upon ligand binding, several conformational changes occur in PfFKBD, including aromatic residues that shape the FK506 binding pocket as shown by NMR studies. A microarray analysis suggests that FK506 and cyclosporine A (CsA) might inhibit parasite development by interfering with the same signaling pathways.

9. Langsley, G., V. van Noort, C. Carret, M. Meissner, E.P. de Villiers, R. Bishop, and A. Pain (2008), Comparative genomics of the Rab protein family in Apicomplexan parasites. inPr_Microbes Infect in Press. PMID: 18468471.

Rab genes encode a subgroup of small GTP-binding proteins within the ras super-family that regulate targeting and fusion of transport vesicles within the secretory and endocytic pathways. These genes are of particular interest in the protozoan phylum Apicomplexa, since a family of Rab GTPases has been described for Plasmodium and most putative secretory pathway proteins in Apicomplexa have conventional predicted signal peptides. Moreover, peptide motifs have now been identified within a large number of secreted Plasmodium proteins that direct their targeting to the red blood cell cytosol, the apicoplast, the food vacuole and Maurer’s clefs; in contrast, motifs that direct proteins to secretory organelles (rhoptries, micronemes and microspheres) have yet to be defined. The nature of the vesicle in which these proteins are transported to their destinations remains unknown and morphological structures equivalent to the endoplasmic reticulum and trans-Golgi stacks typical of other eukaryotes cannot be visualised in Apicomplexa. Since Rab GTPases regulate vesicular traffic in all eukaryotes, and this traffic in intracellular parasites could regulate import of nutrient and drugs and export of antigens, host cell modulatory proteins and lactate we compare and contrast here the Rab families of Apicomplexa.

10. Lazarus, M.D., T.G. Schneider, and T.F. Taraschi (2008), A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum. inPr_J Cell Sci in Press. PMID: 18477610.

The current model for hemoglobin ingestion and transport by intraerythrocytic Plasmodium falciparum malaria parasites shares similarities with endocytosis. However, the model is largely hypothetical, and the mechanisms responsible for the ingestion and transport of host cell hemoglobin to the lysosome-like food vacuole (FV) of the parasite are poorly understood. Because actin dynamics play key roles in vesicle formation and transport in endocytosis, we used the actin-perturbing agents jasplakinolide and cytochalasin D to investigate the role of parasite actin in hemoglobin ingestion and transport to the FV. In addition, we tested the current hemoglobin trafficking model through extensive analysis of serial thin sections of parasitized erythrocytes (PE) by electron microscopy. We find that actin dynamics play multiple, important roles in the hemoglobin transport pathway, and that hemoglobin delivery to the FV via the cytostomes might be required for parasite survival. Evidence is provided for a new model, in which hemoglobin transport to the FV occurs by a vesicle-independent process.

11. Perruchon, J., R. Ortmann, M. Altenkamper, K. Silber, J. Wiesner, H. Jomaa, G. Klebe, and M. Schlitzer (2008), Studies Addressing the Importance of Charge in the Binding of Fosmidomycin-Like Molecules to Deoxyxylulosephosphate Reductoisomerase. inPr_ChemMedChem in Press. PMID: 18470849.

Fosmidomycin and its homologue FR900098 are inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, which is part of the mevalonate-independent isoprenoid biosynthetic pathway. Replacement of the phosphonate moiety by uncharged sulfone or sulfonamide partial structures resulted in complete loss of activity. Dropping one of the two negative charges resulted in a marked decrease in activity. Through occupation of a hydrophobic binding site, some activity could be regained, leading to compounds with micromolar activity against cultured malaria parasites.

12. Rodrigues, J.C. and W. de Souza (2008), Ultrastructural alterations in organelles of parasitic protozoa induced by different classes of metabolic inhibitors. Curr Pharm Des 14(9): 925-38. PMID: 18473841.

Parasitic protozoa such as Leishmania, Trypanosoma, Plasmodium, Toxoplasma gondii, Giardia and Trichomonas are able to cause several diseases affecting millions of people around the world with dramatic consequences to the socio-economic life of the affected countries. Diseases like malaria, leishmaniasis and trypanosomiasis have been classified by the World Health Organization as neglected diseases, because they have been almost completely forgotten by the governments as well as the pharmaceutical companies. The specific chemotherapy currently employed for the treatment of these diseases has serious limitations due to lack of efficacy, toxic side effects, growth of drug-resistance and high costs. Thus, it is urgent to develop new chemotherapeutic agents that are more effective, safe and accessible. In this context, several works have been focused on understanding the effect of different drug-treatments on these parasitic protozoa. Organelles and structures such as mitochondrion, kinetoplast, apicoplast, glycosome, acidocalcisome, hydrogenosome, plasma membrane and the cytoskeleton have been studied using different approaches to identify new targets for the development of new chemotherapeutic agents that are required. Some studies on alterations in the fine structure, as assayed using electron microscopy, have indicated the nature of lesions induced by several drugs, allowing deductions on possible modes of action. Here, we briefly review the available data of the effects of several drugs on the ultrastructure of parasitic protozoa and show how electron microscopy can contribute to elucidate the different mechanisms of these anti-parasitic drugs.

13. Srivastava, A., P. Mukherjee, P.V. Desai, M.A. Avery, and B.L. Tekwani (2008), Structural analysis of farnesyl pyrophosphate synthase from parasitic protozoa, a potential chemotherapeutic target. Infect Disord Drug Targets 8(1): 16-30. PMID: 18473904.

Synthesis of farnesyl pyrophosphate (FPP), a key intermediate of the isoprenoid biosynthesis pathway, is catalyzed by FPP synthase (FPPS). Antiprotozoal properties of bisphosphonates, which target FPPS, have generated interest in FPPS as a potential antiprotozoal drug target. The genes encoding FPPS from parasitic protozoa were assessed to analyze structural and functional features of the enzyme. Comparisons of the FPPS from the parasitic protozoa and search for conserved motifs revealed that FPPS from both apicomplexan and trypanosomatid parasites show characteristic conserved regions for example first aspartate rich motif (FARM) contained within II conserved domain and the second aspartate rich motif (SARM) contained within VI conserved domain. Phylogenetic analysis of FPPS generated a tree with three distinct clusters. Overall topology of the phylogenic tree constructed with small subunit ribosomal RNA sequences was almost similar to that constructed with FPPS sequences. Comparative homology modeling and structural comparisons of FPPS from the parasitic protozoa provided significant insights into common and distinct characteristics of the enzyme. The critical interacting residues of the isopentenyl pyrophosphate binding site are conserved across the enzymes from the family except for malarial FPPS where the C-terminal residues from the BXB motif of helix J were missing. Variations noticed in aromatic residue pairs at the fourth and fifth position upstream of the FARM, which play important role in determination of chain length of the polyprenyl products, may produce functional differences among protozoan FPPSs. The structural comparison of protozoan FPPS may be useful in designing common or selective FPPS inhibitors as potential broad spectrum or selective antiprotozoal agents.

14. Sunil, S., V.S. Chauhan, and P. Malhotra (2008), Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases. inPr_BMC Mol Biol 9_in Press(1): 47. PMID: 18477411.

ABSTRACT: BACKGROUND: Plasmodium falciparum cysteine proteases (falcipains) play indispensable roles in parasite infection and development, especially in the process of host erythrocyte rupture/invasion and hemoglobin degradation. No detailed molecular analysis of transcriptional regulation of parasite proteases especially cysteine proteases has yet been reported. In this study, using a combination of transient transfection assays and electrophoretic mobility shift assays (EMSA), we demonstrate the presence of stage specific nuclear factors that bind to unique sequence elements in the 5′upstream regions of the falcipains and probably modulate the expression of cysteine proteases. RESULTS: Falcipains differ in their timing of expression and exhibit ability to compensate each other’s functions at asexual blood stages of the parasite. Present study was undertaken to study the transcriptional regulation of falcipains. Transient transfection assay employing firefly luciferase as a reporter revealed that a ~1kb sequence upstream of translational start site is sufficient for the functional transcriptional activity of falcipain-1 gene, while falcipain-2, -2′ and -3 genes that exist within 12kb stretch on chromosome 11 require ~2kb upstream sequences for the expression of reporter luciferase activity. EMSA analysis elucidated binding of distinct nuclear factors to specific sequences within the 5′upstream regions of falcipain genes. Analysis of falcipains’ 5′upstream regulatory regions did not reveal the presence of sequences known to bind general eukaryotic factors. However, we did find parasite specific sequence elements such as poly(dA) poly(dT) tracts, CCAAT boxes and a single 7bp-G rich sequence, (A/G)NGGGG(C/A) in the 5′ upstream regulatory regions of these genes, thereby suggesting the role(s) of Plasmodium specific transcriptional factors in the regulation of falcipain genes. CONCLUSIONS: Taken together, these results suggest that expression of Plasmodium cysteine proteases is regulated at the transcriptional level and parasite specific factors regulate the expression of falcipain genes. These findings open new venues for further studies in identification of parasite specific transcription factors.

15. Yoshikawa, M., K. Motoshima, K. Fujimoto, A. Tai, H. Kakuta, and K. Sasaki (2008), Pyridinium cationic-dimer antimalarials, unlike chloroquine, act selectively between the schizont stage and the ring stage of Plasmodium falciparum. inPr_Bioorg Med Chem in Press. PMID: 18479926.

Malaria is a leading cause of death in developing countries, and the emergence of strains resistant to the main therapeutic agent, chloroquine, has become a serious problem. We have developed cationic-dimer type antimalarials, MAP-610 and PMAP-H10, which are structurally different from chloroquine. In this study, we introduced several substituents on the terminal phenyl rings of PMAP-H10. The electronic and hydrophobic properties of the substituents were correlated with the antimalarial activity and cytotoxicity of the compounds, respectively. Studies with synchronized cultures of malarial plasmodia showed that our cationic-dimers act selectively between the schizont stage and the ring stage of the parasitic cycle, unlike chloroquine, which has a stage-independent action. Thus, the mechanism of action of our antimalarials appears to be different from that of chloroquine, and our compounds may be effective against chloroquine-resistant strains.

16. Arechaga, I. and D. Fotiadis (2007), Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis. J Struct Biol 160(3): 287-94. PMID: 17959389.

Mitochondrial F(1)F(o)-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F(1)F(o)-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F(1)F(o)-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F(1)F(o)-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F(1) catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F(1) to the lipid monolayer and the F(o) membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F(o) by electron microscopy and AFM.

17. Lee, I., A.R. Salomon, K. Yu, J.W. Doan, L.I. Grossman, and M. Huttemann (2006), New prospects for an old enzyme: mammalian cytochrome c is tyrosine-phosphorylated in vivo. Biochemistry 45(30): 9121-8. PMID: 16866357.

Mammalian cytochrome c (Cyt c) has two primary functions: transfer of electrons from the bc1 complex to cytochrome c oxidase (COX) as part of the mitochondrial electron transport chain (ETC), and participation in type II apoptosis. Several studies have indicated that components of the ETC can be phosphorylated, and we have recently shown that the Cyt c electron acceptor COX is phosphorylated on Tyr-304 of subunit I in liver upon activation of the cAMP-dependent pathway, leading to strong enzyme inhibition. However, covalent modification of Cyt c through phosphorylation has not yet been reported. We have isolated Cyt c from cow heart under conditions that preserve the physiological in vivo phosphorylation status. Western analysis with an anti-phosphotyrosine antibody indicated tyrosine phosphorylation. The site of phosphorylation was definitively assigned by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS) to Tyr-97, one of the four tyrosine residues present in Cyt c. The phosphorylated tyrosine is part of a motif that contains five residues identical to the tyrosine phosphorylation site in COX subunit I. Spectral analysis revealed that the characteristic 695 nm absorption band is shifted to 687 nm and reversed after treatment with alkaline phosphatase. This band results from the Met-80-heme iron bond, and its shift might indicate changes in the catalytic heme crevice. In vivo phosphorylated Cyt c shows enhanced sigmoidal kinetics with COX, and half-maximal turnover is observed at a Cyt c substrate concentration of 5.5 microM compared to 2.5 microM for alkaline phosphatase-treated Cyt c. Possible consequences of Tyr-97 phosphorylation with respect to cardiolipin binding and of location of Tyr-97 in close proximity to Lys-7, a crucial residue for interaction with Apaf-1 during apoptosis, are discussed.

18. Yamano, K., Y. Yatsukawa, M. Esaki, A.E. Hobbs, R.E. Jensen, and T. Endo (2008), Tom20 and Tom22 share the common signal recognition pathway in mitochondrial protein import. J Biol Chem 283(7): 3799-807. PMID: 18063580.

Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import.

19. Yu, H., I. Lee, A.R. Salomon, K. Yu, and M. Huttemann (2008), Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration. inPr_Biochim Biophys Acta in Press. PMID: 18471988.

Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc(1) complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s(-1) whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s(-1). Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.

[1-10]

Other Science.

[11-16]

Annotated Citations.

[note: “in Press”, “online advance publication”, etc indicated by “inPr_” preceeding journal name. “Final_” indicates a final version of an article previously downloaded in an “inPr_” format. “NA_” indicates journals for which the Library system does not have an electronic subscription (as far as I can tell).]

1. Ayi, K., G. Min-Oo, L. Serghides, M. Crockett, M. Kirby-Allen, I. Quirt, P. Gros, and K.C. Kain (2008), Pyruvate kinase deficiency and malaria. N Engl J Med 358(17): 1805-10. PMID: 18420493.

Malaria that is caused by Plasmodium falciparum is a significant global health problem. Genetic characteristics of the host influence the severity of disease and the ultimate outcome of infection, and there is evidence of coevolution of the plasmodium parasite with its host. In humans, pyruvate kinase deficiency is the second most common erythrocyte enzyme disorder. Here, we show that pyruvate kinase deficiency provides protection against infection and replication of P. falciparum in human erythrocytes, raising the possibility that mutant pyruvate kinase alleles may confer a protective advantage against malaria in human populations in areas where the disease is endemic.

2. Biagini, G.A., N. Fisher, N. Berry, P.A. Stocks, B. Meunier, D.P. Williams, R. Bonar-Law, P.G. Bray, A. Owen, P.M. O’Neill, and S.A. Ward (2008), Acridinediones: selective and potent inhibitors of the malaria parasite mitochondrial bc1 complex. Final_Mol Pharmacol 73(5): 1347-55. PMID: 18319379.

The development of drug resistance to affordable drugs has contributed to a global increase in the number of deaths from malaria. This unacceptable situation has stimulated research for new drugs active against multidrug-resistant Plasmodium falciparum parasites. In this regard, we show here that deshydroxy-1-imino derivatives of acridine (i.e., dihydroacridinediones) are selective antimalarial drugs acting as potent (nanomolar K(i)) inhibitors of parasite mitochondrial bc(1) complex. Inhibition of the bc(1) complex led to a collapse of the mitochondrial membrane potential, resulting in cell death (IC(50) approximately 15 nM). The selectivity of one of the dihydroacridinediones against the parasite enzyme was some 5000-fold higher than for the human bc(1) complex, significantly higher ( approximately 200 fold) than that observed with atovaquone, a licensed bc(1)-specific antimalarial drug. Experiments performed with yeast manifesting mutations in the bc(1) complex reveal that binding is directed to the quinol oxidation site (Q(o)) of the bc(1) complex. This is supported by favorable binding energies for in silico docking of dihydroacridinediones to P. falciparum bc(1) Q(o). Dihydroacridinediones represent an entirely new class of bc(1) inhibitors and the potential of these compounds as novel antimalarial drugs is discussed.

3. Chung, M.C., E.I. Ferreira, J.L. Santos, J. Giarolla, D.G. Rando, A.E. Almeida, P.L. Bosquesi, R.F. Menegon, and L. Blau (2008), Prodrugs for the treatment of neglected diseases. Molecules 13(3): 616-77. PMID: 18463559.

Recently, World Health Organization (WHO) and Medicins San Frontieres (MSF) proposed a classification of diseases as global, neglected and extremely neglected.Global diseases, such as cancer, cardiovascular and mental (CNS) diseases represent the targets of the majority of the R&D efforts of pharmaceutical companies. Neglected diseases affect millions of people in the world yet existing drug therapy is limited and often inappropriate. Furthermore, extremely neglected diseases affect people living under miserable conditions who barely have access to the bare necessities for survival. Most of these diseases are excluded from the goals of the R&D programs in the pharmaceutical industry and therefore fall outside the pharmaceutical market. About 14 million people,mainly in developing countries, die each year from infectious diseases. From 1975 to 1999,1393 new drugs were approved yet only 1% were for the treatment of neglected diseases[3]. These numbers have not changed until now, so in those countries there is an urgent need for the design and synthesis of new drugs and in this area the prodrug approach is a very interesting field. It provides, among other effects, activity improvements and toxicity decreases for current and new drugs, improving market availability. It is worth noting that it is essential in drug design to save time and money, and prodrug approaches can be considered of high interest in this respect. The present review covers 20 years of research on the design of prodrugs for the treatment of neglected and extremely neglected diseases such as Chagas’ disease (American trypanosomiasis), sleeping sickness (African trypanosomiasis), malaria, sickle cell disease, tuberculosis, leishmaniasis and schistosomiasis.

4. Durand, P.M. and T.L. Coetzer (2008), Pyruvate kinase deficiency protects against malaria in humans. inPr_Haematologica in Press. PMID: 18460648.

The association between inherited erythrocyte disorders in humans and resistance to malaria is well documented and includes enzyme deficiencies, hemoglobinopathies and membrane protein abnormalities. The association between pyruvate kinase (PK) deficiency and resistance to malaria has been demonstrated in vivo using a murine model1 and it has been suggested that this phenomenon may extend to humans. The data presented in this paper demonstrate for the first time that PK deficient human erythrocytes are resistant to malaria infection in vitro, and support the in vivo findings in the murine model.

5. Joshi, S., A.R. Singh, A. Kumar, P.C. Misra, M.I. Siddiqi, and J.K. Saxena (2008), Molecular cloning and characterization of Plasmodium falciparum transketolase. inPr_Mol Biochem Parasitol in Press. PMID: 18456347.

The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K(m)(app) values of 2.25 and 4.78mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K(i) value of 305muM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% alpha-helices and 26% beta-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.

6. Kato, N., T. Sakata, G. Breton, K.G. Le Roch, A. Nagle, C. Andersen, B. Bursulaya, K. Henson, J. Johnson, K.A. Kumar, F. Marr, D. Mason, C. McNamara, D. Plouffe, V. Ramachandran, M. Spooner, T. Tuntland, Y. Zhou, E.C. Peters, A. Chatterjee, P.G. Schultz, G.E. Ward, N. Gray, J. Harper, and E.A. Winzeler (2008), Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility. inPr_Nat Chem Biol in Press. PMID: 18454143.

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.

7. Koka, S., C. Lang, O.M. Niemoeller, K.M. Boini, J.P. Nicolay, S.M. Huber, and F. Lang (2008), Influence of NO Synthase Inhibitor L-NAME on Parasitemia and Survival of Plasmodium berghei Infected Mice. Cell Physiol Biochem 21(5-6): 481-488. PMID: 18453756.

Accelerated suicidal death or eryptosis of infected erythrocytes may delay development of parasitemia in malaria. Eryptosis is inhibited by nitric oxide (NO). The present study has been performed to explore, whether inhibition of NO synthase by L-NAME modifies the course of malaria. We show here that L-NAME (>10 muM) increased phosphatidylserine exposure of Plasmodium falciparum infected human erythrocytes, an effect significantly more marked than in noninfected human erythrocytes. We further show that parasitemia in Plasmodium berghei infected mice was significantly decreased (from 50% to 18% of circulating erythrocytes 20 days after infection) by addition of 1 mg/ml L-NAME to the drinking water. According to CFSE labelling L-NAME treatment accelerated the clearance of both, noninfected and infected, erythrocytes from circulating blood, but did not significantly extend the life span of infected animals. In conclusion, treatment with L-NAME shortens the life span of circulating erythrocytes and thus delays development of parasitemia during malaria.

8. Korde, R., A. Bhardwaj, R. Singh, A. Srivastava, V.S. Chauhan, R.K. Bhatnagar, and P. Malhotra (2008), A Prodomain Peptide of Plasmodium falciparum Cysteine Protease (Falcipain-2) Inhibits Malaria Parasite Development. inPr_J Med Chem in Press. PMID: 18461922.

Falcipain-2 (FP-2), a papain family cysteine protease of Plasmodium falciparum, is a promising target for antimalarial chemotherapy. Designing inhibitors that are highly selective for falcipain-2 has been difficult because of broad specificity of different cysteine proteinases. Because propeptide regions of cysteine proteases have been shown to inhibit their cognate enzymes specifically and selectively, in the present study, we evaluated the inhibitory potential of few falcipain-2 proregion peptides. A 15 residue peptide (PP1) inhibited falcipain-2 enzyme activity in vitro. Studies on the uptake of PP1 into the parasitized erythrocytes showed access of peptide into the infected RBCs. PP1 fused with Antennapedia homeoprotein internalization domain blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth and maturation. Together, our results identify a peptide derived from the proregion of falcipain-2 that blocks late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric drugs against the human malaria parasite.

9. Muller, I.B., R. Das Gupta, K. Luersen, C. Wrenger, and R.D. Walter (2008), Assessing the polyamine metabolism of Plasmodium falciparum as chemotherapeutic target. inPr_Mol Biochem Parasitol in Press. PMID: 18455248.

More than 30 years ago the potent ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) was designed as new anticancer drug. Its efficacy was not as expected since the polyamine metabolism in mammalian cells seemed to be far more complex. However when DFMO was applied to African trypanosomes its effect on this protozoan parasite was highly convincing. Thenceforward many researchers tested DFMO and also other polyamine synthesis inhibitors against different parasites among them the causative agent of malaria Plasmodium. This review recapitulates the different attempts to interfere chemically with the plasmodial polyamine metabolism, the impact on the disease as well as its biochemical and molecular background. It will show that this fast proliferating organism depends for growth on high amounts of polyamines and that Plasmodium has its own and unique polyamine synthesis, differing highly from the mammalian one mainly in the arrangement of the key enzymes, S-adenosylmethionine decarboxylase and ornithine decarboxylase (AdoMetDC/ODC), on a bifunctional protein.

10. Oleinikov, A.V., S.E. Francis, J.R. Dorfman, E. Rossnagle, S. Balcaitis, T. Getz, M. Avril, S. Gose, J.D. Smith, M. Fried, and P.E. Duffy (2008), VAR2CSA Domains Expressed in Escherichia coli Induce Cross-Reactive Antibodies to Native Protein. J Infect Dis 197(8): 1119-23. PMID: 18462161.

The variant surface antigen VAR2CSA is a pregnancy malaria vaccine candidate, but its size and polymorphism are obstacles to development. We expressed 3D7-type VAR2CSA domains in Escherichia coli as insoluble His-tagged proteins (Duffy binding-like [DBL] domains DBL1, DBL3, DBL4, and DBL5) that were denatured and refolded or as soluble glutathione S-transferase-tagged protein (DBL6). Anti-DBL5 antiserum cross-reacted with surface proteins of chondroitin sulfate A (CSA)-binding laboratory strains (3D7-CSA and FCR3-CSA) and a clinical pregnancy malaria isolate, whereas anti-DBL6 antiserum reacted only to 3D7 surface protein. This is the first report that E. coli-expressed VAR2CSA domains induce antibody to native VAR2CSA.

11. Barnard, E., N.V. McFerran, A. Trudgett, J. Nelson, and D.J. Timson (2008), Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method. Fungal Genet Biol 45(5): 597-604. PMID: 18313953.

An alternative method for monitoring protein-protein interactions in Saccharomyces cerevisiae has been developed. It relies on the ability of two fragments of enhanced green fluorescent protein (EGFP) to reassemble and fluoresce when fused to interacting proteins. Since this fluorescence can be detected in living cells, simultaneous detection and localisation of interacting pairs is possible. DNA sequences encoding N- and C-terminal EGFP fragments flanked by sequences from the genes of interest were transformed into S. cerevisiae JPY5 cells and homologous recombination into the genome verified by PCR. The system was evaluated by testing known interacting proteins: labelling of the phosphofructokinase subunits, Pfk1p and Pfk2p, with N- and C-terminal EGFP fragments, respectively, resulted in green fluorescence in the cytoplasm. The system works in other cellular compartments: labelling of Idh1p and Idh2p (mitochondrial matrix), Sdh3p and Sdh4p (mitochondrial membrane) and Pap2p and Mtr4p (nucleus) all resulted in fluorescence in the appropriate cellular compartment.

12. Buch-Pedersen, M.J., B.P. Pedersen, B. Veierskov, P. Nissen, and M.G. Palmgren (2008), Protons and how they are transported by proton pumps. inPr_Pflugers Arch in Press. PMID: 18458946.

The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded ATPases extrude protons from cells of plants and fungi to generate electrochemical proton gradients. The recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Taking the biochemical and structural data together, we are now able to describe the basic molecular components that allow the plasma membrane proton H(+)-ATPase to carry out proton transport against large membrane potentials. When divergent proton pumps such as the plasma membrane H(+)-ATPase, bacteriorhodopsin, and F(O)F(1) ATP synthase are compared, unifying mechanistic premises for biological proton pumps emerge. Most notably, the minimal pumping apparatus of all pumps consists of a central proton acceptor/donor, a positively charged residue to control pK (a) changes of the proton acceptor/donor, and bound water molecules to facilitate rapid proton transport along proton wires.

13. Morth, J.P., B.P. Pedersen, M.S. Toustrup-Jensen, T.L. Sorensen, J. Petersen, J.P. Andersen, B. Vilsen, and P. Nissen (2007), Crystal structure of the sodium-potassium pump. Nature 450(7172): 1043-9. PMID: 18075585.

The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the alpha-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The beta- and gamma-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices alphaM7/alphaM10 and alphaM9, respectively. The gamma-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the alpha-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.

14. Olesen, C., M. Picard, A.M. Winther, C. Gyrup, J.P. Morth, C. Oxvig, J.V. Moller, and P. Nissen (2007), The structural basis of calcium transport by the calcium pump. Nature 450(7172): 1036-42. PMID: 18075584.

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.

15. Pedersen, B.P., M.J. Buch-Pedersen, J.P. Morth, M.G. Palmgren, and P. Nissen (2007), Crystal structure of the plasma membrane proton pump. Nature 450(7172): 1111-4. PMID: 18075595.

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.

16. Thomas, D., P. Bron, T. Weimann, A. Dautant, M.F. Giraud, P. Paumard, B. Salin, A. Cavalier, J. Velours, and D. Brethes (2008), Supramolecular organization of the yeast F 1F o-ATP synthase. inPr_Biol Cell in Press. PMID: 18447829.

Background information. The yeast mitochondrial F 1F o-ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyze ATP synthesis from ADP and Pi. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology. Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze fracture, negative staining and image processing we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions. Our results shed new light on the supramolecular organization of the F 1F o-ATP synthase and its potential role in mitochondrial morphology.

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