Patterns of selection on Plasmodium falciparum erythrocyte‐binding antigens after the colonization of the New World

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte‐binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba‐140, eba‐175, eba‐181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba‐140 seemed to be under stronger diversifying selection in South America than eba‐175. In contrast, eba‐181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins.     

The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum

It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.     

Genomewide identification and analysis of heat‐shock proteins 70/110 to reveal their potential functions in Chinese soft‐shelled turtle Pelodiscus sinensis

Heat‐shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft‐shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved “heat shock protein 70” domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue‐specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B‐L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.     

Co‐evolution‐driven switch of J‐protein specificity towards an Hsp70 partner

Molecular mechanisms by which protein–protein interactions are preserved or lost after gene duplication are not understood. Taking advantage of the well–studied yeast mtHsp70:J–protein molecular chaperone system, we considered whether changes in partner proteins accompanied specialization of gene duplicates. Here, we report that existence of the Hsp70 Ssq1, which arose by duplication of the gene encoding multifunction mtHsp70 and specializes in iron–sulphur cluster biogenesis, correlates with functional and structural changes in the J domain of its J–protein partner Jac1. All species encoding this shorter alternative version of the J domain share a common ancestry, suggesting that all short JAC1 proteins arose from a single deletion event. Construction of a variant that extended the length of the J domain of a ‘short’ Jac1 enhanced its ability to partner with multifunctional Hsp70. Our data provide a causal link between changes in the J protein partner and specialization of duplicate Hsp70.     

Co‐evolutionary patterns and diversification of ant–fungus associations in the asexual fungus‐farming ant Mycocepurus smithii in Panama

Partner fidelity through vertical symbiont transmission is thought to be the primary mechanism stabilizing cooperation in the mutualism between fungus‐farming (attine) ants and their cultivated fungal symbionts. An alternate or additional mechanism could be adaptive partner or symbiont choice mediating horizontal cultivar transmission or de novo domestication of free‐living fungi. Using microsatellite genotyping for the attine ant Mycocepurus smithii and ITS rDNA sequencing for fungal cultivars, we provide the first detailed population genetic analysis of local ant–fungus associations to test for the relative importance of vertical vs. horizontal transmission in a single attine species. M. smithii is the only known asexual attine ant, and it is furthermore exceptional because it cultivates a far greater cultivar diversity than any other attine ant. Cultivar switching could permit the ants to re‐acquire cultivars after garden loss, to purge inferior cultivars that are locally mal‐adapted or that accumulated deleterious mutations under long‐term asexuality. Compared to other attine ants, symbiont choice and local adaptation of ant–fungus combinations may play a more important role than partner‐fidelity feedback in the co‐evolutionary process of M. smithii and its fungal symbionts.     

Sorghum bicolor SbHSP110 has an elongated shape and is able of protecting against aggregation and replacing human HSPH1/HSP110 in refolding and disaggregation assays

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.     

Hsp90 and co‐chaperones twist the functions of diverse client proteins

Hsp90 molecular chaperones are required for the stability and activity of a diverse range of client proteins that have critical roles in signal transduction, cellular trafficking, chromatin remodeling, cell growth, differentiation, and reproduction. Mammalian cells contain three types of Hsp90s: cytosolic Hsp90, mitochondrial Trap‐1, and Grp94 of the endoplasmic reticulum. Each of the Hsp90s, as well as the bacterial homolog, HtpG, hydrolyze ATP and undergo similar conformational changes. Unlike the other forms of Hsp90, cytosolic Hsp90 function is dependent on a battery of co‐chaperone proteins that regulate the ATPase activity of Hsp90 or direct Hsp90 to interact with specific client proteins. This review will summarize what is known about Hsp90's ability to mediate the folding and activation of diverse client proteins that contribute to human diseases, such as cancer and fungal and viral infections. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 211–217, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

On the evolutionary origin of the chaperonins

An analysis of the apical domain of the Group-I and Group-II chaperonins shows that they have structural similarities to two different protein folds: a "swivel-domain" phosphotransferase and a thioredoxin-like peroxiredoxin. There is no significant sequence similarity that supports either similarity and the degree of similarity based on structure is comparable but weak for both relationships. Based on possible evolutionary transitions, we deduced that a phosphotransferase origin would require both a large insertion and deletion of structure whereas a peroxiredoxin origin requires only a peripheral rearrangement, similar to an internal domain-swap. We postulate that this change could have been triggered by the insertion of a peroxiredoxin into the ATPase domain that led to the modern chaperonin domain arrangement. The peroxidoxin fold is the most highly embellished member of the thioredoxin super-family and the insertion event may have "overloaded" the core, leading to a rearrangement. A peroxiredoxin origin for the domain also provides a functional explanation, as the peroxiredoxins can act as chaperones when they adopt a multimeric ring complex, similar to the chaperonin subunit configuration. In addition, several of the GroEL apical domain hydrophobic residues which interact with the unfolded protein are located in a position that corresponds to the protein substrate binding region of the peroxiredoxin fold. We suggest that the origin of the ur-chaperonin from a thioredoxin/peroxiredoxin fold might also account for the number of thioredoxin-fold containing proteins that interact with chaperonins, such as tubulin and phosducin-like proteins.     

Functional and evolutionary analysis of viral proteins containing a Rossmann‐like fold

Viruses are the most abundant life form and infect practically all organisms. Consequently, these obligate parasites are a major cause of human suffering and economic loss. Rossmann‐like fold is the most populated fold among α/β‐folds in the Protein Data Bank and proteins containing Rossmann‐like fold constitute 22% of all known proteins 3D structures. Thus, analysis of viral proteins containing Rossmann‐like domains could provide an understanding of viral biology and evolution as well as could propose possible targets for antiviral therapy. We provide functional and evolutionary analysis of viral proteins containing a Rossmann‐like fold found in the evolutionary classification of protein domains (ECOD) database developed in our lab. We identified 81 protein families of bacterial, archeal, and eukaryotic viruses in light of their evolution‐based ECOD classification and Pfam taxonomy. We defined their functional significance using enzymatic EC number assignments as well as domain‐level family annotations.     

The use of stress‐70 proteins in physiology: a re‐appraisal

There are few factors more important to the mechanisms of evolution than stress. The stress response has formed as a result of natural selection, improving the capacity of organisms to withstand situations that require action. The ubiquity of the cellular stress response suggests that effective mechanisms to counteract stress emerged early in the history of life, and their commonality proves how vital such mechanisms are to operative evolution. The cellular stress response (CSR) has been identified as a characteristic of cells in all three domains of life and consists of a core 44 proteins that are structurally highly conserved and that have been termed the ‘minimal stress proteome’ (MSP). Within the MSP, the most intensely researched proteins are a family of heat‐shock proteins known as HSP70. Superficially, correlations between the induction of stress and HSP70 differential expression support the use of HSP70 expression as a nonspecific biomarker of stress. However, we argue that too often authors have failed to question exactly what HSP70 differential expression signifies. Herein, we argue that HSP70 up‐regulation in response to stressors has been shown to be far more complex than the commonly accepted quasi‐linear relationship. In addition, in many instances, the uncertain identity and function of heat‐shock proteins and heat‐shock cognates has led to difficulties in interpretation of reports of inducible heat‐shock proteins and constitutive heat‐shock cognates. We caution against the broad application of HSP70 as a biomarker of stress in isolation and conclude that the application of HSP70 as a meaningful index of stress requires a higher degree of validation than the majority of research currently undertakes.     

Co‐evolution of gametes of the Greater Bandicoot Rat,Bandicota indica – a murine rodent from South‐East Asia

Most Old World mice and rats, subfamily Murinae, have a spermatozoon with an apical hook, a long tail and, as seen typically in eutherian mammals, a bilaterally flattened head. Dramatically different from this are the sperm of the Greater Bandicoot Rat, Bandicota indica. Here, we ask the question has the structure of the sperm head co‐evolved with that of the egg coat, the zona pellucida? For this, we first summarise the morphological features of the spermatozoon of B. indica that may relate to zona pellucida penetration at the time of fertilisation, and we confirm that the sperm head is generally round, not bilaterally flattened, in profile and has a huge acrosome. We then show that the zona pellucida around oocytes in tertiary follicles also differs from that of the other murine rodents in being only about 4 μm thick and, as demonstrated by lectin staining, has an unusual abundance of alpha‐L‐fucose. These findings indicate that both the male and female gametes of this South‐East Asian murine rodent are highly divergent in their structural organisation. One of the functional implications of this probably relates to sperm–zona interactions and the release of acrosomal enzymes that probably facilitate penetration by digestion of the zona matrix at the time of fertilisation.     

Right‐ and left‐handed three‐helix proteins. I. Experimental and simulation analysis of differences in folding and structure

Despite the large number of publications on three‐helix protein folding, there is no study devoted to the influence of handedness on the rate of three‐helix protein folding. From the experimental studies, we make a conclusion that the left‐handed three‐helix proteins fold faster than the right‐handed ones. What may explain this difference? An important question arising in this paper is whether the modeling of protein folding can catch the difference between the protein folding rates of proteins with similar structures but with different folding mechanisms. To answer this question, the folding of eight three‐helix proteins (four right‐handed and four left‐handed), which are similar in size, was modeled using the Monte Carlo and dynamic programming methods. The studies allowed us to determine the orders of folding of the secondary‐structure elements in these domains and amino acid residues which are important for the folding. The obtained data are in good correlation with each other and with the experimental data. Structural analysis of these proteins demonstrated that the left‐handed domains have a lesser number of contacts per residue and a smaller radius of cross section than the right‐handed domains. This may be one of the explanations of the observed fact. The same tendency is observed for the large dataset consisting of 332 three‐helix proteins (238 right‐ and 94 left‐handed). From our analysis, we found that the left‐handed three‐helix proteins have some less‐dense packing that should result in faster folding for some proteins as compared to the case of right‐handed proteins.Proteins 2013; © 2013 Wiley Periodicals, Inc.     

The evolution and evolutionary consequences of marginal thermostability in proteins

When we seek to explain the characteristics of living systems in their evolutionary context, we are often interested in understanding how and why certain properties arose through evolution, and how these properties then affected the continuing evolutionary process. This endeavor has been assisted by the use of simple computational models that have properties characteristic of natural living systems but allow simulations over evolutionary timescales with full transparency. We examine a model of the evolution of a gene under selective pressure to code for a protein that exists in a prespecified folded state at a given growth temperature. We observe the emergence of proteins with modest stabilities far below those possible with the model, with a denaturation temperature tracking the simulation temperature, despite the absence of selective pressure for such marginal stability. This demonstrates that neither observations of marginally stable proteins, nor even instances where increased stability interferes with function, provide evidence that marginal stability is an adaptation. Instead the marginal stability is the result of a balance between predominantly destabilizing mutations and selection that shifts depending on effective population size. Even if marginal stability is not an adaptation, the natural tendency of proteins toward marginal stability, and the range of stabilities that occur during evolution, may have significant effect on the evolutionary process.     

Characterization and evolutionary analysis of tributyltin‐binding protein and pufferfish saxitoxin and tetrodotoxin‐binding protein genes in toxic and nontoxic pufferfishes

Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long‐standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin‐binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin‐binding protein type 2 (TBT‐bp2) domains. In this study, we examined the evolutionary dynamics of TBT‐bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR‐based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT‐bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT‐bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT‐bp2 genes. In addition, a detailed comparison of Takifugu TBT‐bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT‐bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.     

Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria

Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M. bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.     

分子伴侣的功能和应用

本文综述了分子伴侣的分类、功能、作用机理、研究现状及应用前景。分子伴侣是在生物大分子的折叠、组装、转运及降解等过程中起协助作用,参与协助抗原的呈递和遗传物质的复制、转录及构象的确立,但自身并不发生任何变化的一大类广泛存在于生物体内的蛋白质分子。随着对分子伴侣的进一步研究和相关知识的不断深入,分子伴侣在生物产品开发、物种改良、抗衰老,疾病预防、诊断和治疗以及环境监测方面具有广阔的前景。     

Chaperonin 60: a paradoxical,evolutionarily conserved protein family with multiple moonlighting functions

Chaperonin 60 is the prototypic molecular chaperone, an essential protein in eukaryotes and prokaryotes, whose sequence conservation provides an excellent basis for phylogenetic analysis. Escherichia coli chaperonin 60 (GroEL), the prototype of this family of proteins, has an established oligomeric‐structure‐based folding mechanism and a defined population of folding partners. However, there is a growing number of examples of chaperonin 60 proteins whose crystal structures and oligomeric composition are at variance with GroEL, suggesting that additional complexities in the protein‐folding function of this protein should be expected. In addition, many organisms have multiple chaperonin 60 proteins, some of which have lost their protein‐folding ability. It is emerging that this highly conserved protein has evolved a bewildering variety of additional biological functions – known as moonlighting functions – both within the cell and in the extracellular milieu. Indeed, in some organisms, it is these moonlighting functions that have been left after the loss of the protein‐folding activity. This highlights the major paradox in the biology of chaperonin 60. This article reviews the relationship between the folding and non‐folding (moonlighting) activities of the chaperonin 60 family and discusses current knowledge on their molecular evolution focusing on protein domains involved in the non‐folding chaperonin functions in an attempt to understand the emerging biology of this evolutionarily ancient protein family.     

Dynamics of Plasmodium falciparum enoyl‐ACP reductase and implications on drug discovery

Enoyl‐acyl carrier protein reductase (ENR) is a crucial enzyme in the type II fatty acid synthesis pathway of many pathogens such as Plasmodium falciparum, the etiological agent of the most severe form of malaria. Because of its essential function of fatty acid double bond reduction and the absence of a human homologue, PfENR is an interesting drug target. Although extensive knowledge of the protein structure has been gathered over the last decade, comparatively little remains known about the dynamics of this crucial enzyme. Here, we perform extensive molecular dynamics simulations of tetrameric PfENR in different states of cofactor and ligand binding, and with a variety of different ligands bound. A pocket‐volume analysis is also performed, and virtual screening is used to identify potential druggable hotspots. The implications of the results for future drug‐discovery projects are discussed.     

Trafficking of PfExp1 to the parasitophorous vacuolar membrane of Plasmodium falciparum is independent of protein folding and the PTEX translocon

Having entered the mature human erythrocyte, the malaria parasite survives and propagates within a parasitophorous vacuole, a membrane‐bound compartment separating the parasite from the host cell cytosol. The bounding membrane of this vacuole, referred to as the parasitophorous vacuolar membrane (PVM), contains parasite‐encoded proteins, but how these membrane proteins are trafficked to the PVM remains unknown. Here, we have studied the trafficking of PfExp1 to the PVM. We find that trafficking of PfExp1 to the PVM is independent of the folding state of the protein and also continues unabated upon inactivation of the PVM translocon Plasmodium Translocon of Exported proteins (PTEX). Our data strongly suggest that the trafficking of membrane proteins to the PVM occurs by as yet unknown mechanism, potentially unique to Plasmodium.