The structural basis for CD36 binding by the malaria parasite

CD36 is a scavenger receptor involved in fatty acid metabolism, innate immunity and angiogenesis. It interacts with lipoprotein particles and facilitates uptake of long chain fatty acids. It is also the most common target of the PfEMP1 proteins of the malaria parasite, Plasmodium falciparum, tethering parasite-infected erythrocytes to endothelial receptors. This prevents their destruction by splenic clearance and allows increased parasitaemia. Here we describe the structure of CD36 in complex with long chain fatty acids and a CD36-binding PfEMP1 protein domain. A conserved hydrophobic pocket allows the hugely diverse PfEMP1 protein family to bind to a conserved phenylalanine residue at the membrane distal tip of CD36. This phenylalanine is also required for CD36 to interact with lipoprotein particles. By targeting a site on CD36 that is required for its physiological function, PfEMP1 proteins maintain the ability to tether to the endothelium and avoid splenic clearance

Women with endometriosis have higher comorbidities: Analysis of domestic data in Taiwan

AbstractEndometriosis, defined by the presence of viable extrauterine endometrial glands and stroma, can grow or bleed cyclically, and possesses characteristics including a destructive, invasive, and metastatic nature. Since endometriosis may result in pelvic inflammation, adhesion, chronic pain, and infertility, and can progress to biologically malignant tumors, it is a long-term major health issue in women of reproductive age. In this review, we analyze the Taiwan domestic research addressing associations between endometriosis and other diseases. Concerning malignant tumors, we identified four studies on the links between endometriosis and ovarian cancer, one on breast cancer, two on endometrial cancer, one on colorectal cancer, and one on other malignancies, as well as one on associations between endometriosis and irritable bowel syndrome, one on links with migraine headache, three on links with pelvic inflammatory diseases, four on links with infertility, four on links with obesity, four on links with chronic liver disease, four on links with rheumatoid arthritis, four on links with chronic renal disease, five on links with diabetes mellitus, and five on links with cardiovascular diseases (hypertension, hyperlipidemia, etc.). The data available to date support that women with endometriosis might be at risk of some chronic illnesses and certain malignancies, although we consider the evidence for some comorbidities to be of low quality, for example, the association between colon cancer and adenomyosis/endometriosis. We still believe that the risk of comorbidity might be higher in women with endometriosis than that we supposed before. More research is needed to determine whether women with endometriosis are really at risk of these comorbidities

Patriotic Fun: Toys and Mobilization in China from the Republican to the Communist Era

This chapter explores the use of leisure to mobilize children in China from the 1910s to the early 1950s, in times of both war and peace. Drawing on normative advice, and commenting on youngsters’ reactions, it describes how ostensibly different regimes similarly deployed toys and play in order to foster children’s engagement in struggles of a political, commercial or military nature. It outlines how a variety of items - from so-called “educational” war toys to figurines and lanterns - could serve to rally children for the nation and familiarize war. The chapter argues that, although mobilization was construed as defensive, patriotic activism and acquaintance with the metaphorical or real battlefield were significant components of Chinese children’s upbringing from the beginning of the twentieth century

Insulin-Like Growth Factor-Binding Proteins of Teleost Fishes

The insulin-like growth factor (Igf) binding protein (Igfbp) family has a broad range of physiological functions and a fascinating evolutionary history. This review focuses on the Igfbps of teleost fishes, where genome duplication events have diversified gene repertoire, function, and physiological regulation—with six core Igfbps expanded into a family of over twenty genes in some lineages. In addition to briefly summarizing the current state of knowledge on teleost Igfbp evolution, function, and expression-level regulation, we highlight gaps in our understanding and promising areas for future work

Structural studies of PfEMP1 bound to human cluster of differentiation 36 and of an unusual human antibody

In Plasmodium falciparum-infected humans, parasites escape from splenic clearance and cause severe or chronic symptoms partly through the action of the variable surface antigens expressed on the surfaces of infected erythrocytes, called P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 that interact with human Cluster of Differentiation 36 (CD36) receptor mediate the most common adhesion phenotype found in infected erythrocytes. Here the first crystal structure of CD36 in complex with a PfEMP1 CIDR domain is presented. Together with sequence analysis and biophysical data, it shows how parasite PfEMP1 proteins are able to diversify hugely while retaining a chemically conserved CD36 binding surface. This reveals insight into the structure and function of CD36 and shows how the parasite targets a region of CD36 that is critical for its physiological role, avoiding mutations in host receptors that would prevent parasite binding. Repetitive interspersed family proteins (RIFINs) are the most abundant variant surface antigens in P. falciparum. However, unlike the well-studied PfEMP1, the function of RIFIN remains unclear. Recently, a subset of human antibodies was identified from malaria-endemic areas that can recognise varied RIFINs. These antibodies have the unique feature of the insertion of a human receptor, LAIR1 (CD305), into the third complementarity-determining region of their heavy chain. Here, the first structure of one of these unusual LAIR1-containing antibodies is presented. It reveals that the CDR loops are mostly occluded by the insert and form a surface that positions LAIR1 for antigen binding. The variable regions of the antibody therefore no longer perform their classical role in antigen recognition. Instead they act as an adaptor between a human LAIR1 insert with antigen binding properties and the immune-modulatory antibody constant regions.</p

Structural studies of PfEMP1 bound to human cluster of differentiation 36 and of an unusual human antibody

In Plasmodium falciparum-infected humans, parasites escape from splenic clearance and cause severe or chronic symptoms partly through the action of the variable surface antigens expressed on the surfaces of infected erythrocytes, called P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 that interact with human Cluster of Differentiation 36 (CD36) receptor mediate the most common adhesion phenotype found in infected erythrocytes. Here the first crystal structure of CD36 in complex with a PfEMP1 CIDR domain is presented. Together with sequence analysis and biophysical data, it shows how parasite PfEMP1 proteins are able to diversify hugely while retaining a chemically conserved CD36 binding surface. This reveals insight into the structure and function of CD36 and shows how the parasite targets a region of CD36 that is critical for its physiological role, avoiding mutations in host receptors that would prevent parasite binding. Repetitive interspersed family proteins (RIFINs) are the most abundant variant surface antigens in P. falciparum. However, unlike the well-studied PfEMP1, the function of RIFIN remains unclear. Recently, a subset of human antibodies was identified from malaria-endemic areas that can recognise varied RIFINs. These antibodies have the unique feature of the insertion of a human receptor, LAIR1 (CD305), into the third complementarity-determining region of their heavy chain. Here, the first structure of one of these unusual LAIR1-containing antibodies is presented. It reveals that the CDR loops are mostly occluded by the insert and form a surface that positions LAIR1 for antigen binding. The variable regions of the antibody therefore no longer perform their classical role in antigen recognition. Instead they act as an adaptor between a human LAIR1 insert with antigen binding properties and the immune-modulatory antibody constant regions.</p

Structural and Functional Studies of Heterotetrameric Geranyl Pyrophosphate Synthase from Mentha x piperita

類異戊二烯族 (isoprenoid)為廣泛分布於自然界中的化合物，是以異戊二烯焦磷酸 (isopentenyl pyrophosphate，IPP)為骨架，利用類異戊二烯轉移酵素 (prenyltransferase)催化合成的聚合物。這些聚合物在生物體內扮演不同重要的生理角色，包括構成膜細胞的骨架，參與電子傳遞及訊息傳導，醣蛋白的合成，及蛋白質後修飾作用等等，因此，此類酵素必須非常精確調控其催化反應。根據文獻指出，針對此類酵素發展出之抑制劑也被運用為治療癌症及各種疾病的藥物，例如泰克索 (taxol)及類視色素 (retinoids)可用來抑制癌細胞的生長及誘導細胞走向細胞凋亡;斯他丁 (statin)可用於協助膽固醇過高或有心血管疾病的患者降低膽固醇和心血管疾病風險。近年來，利用X光立體結構與定點突變，對合成15到40碳數之類異戊二烯轉移酵素 (例如，法呢基焦磷酸合成酵素 (farnesyl pyrophosphate synthase，FPPs)，四異戊二烯焦磷酸合成酵素 (granylgeranyl pyrophosphate synthase，GGPPs)，六異戊二烯焦磷酸合成酵素 (hexaprenyl pyrophosphate synthase，HexPPs)，八異戊二烯焦磷酸合成酵素 (octaprenyl pyrophosphate synthase，OPPs)的調控及機制都有深入的探討與了解。然而，直到今日，對此類酵素的立體結構卻仍侷限於兩個相同單元體 (monomer)所組成的”同型”二聚體 (homodimer)。根據文獻指出，有部分的類異戊二烯轉移酵素是必須利用不同的單元體組成”異型”二聚體或四聚體 (heterodimer or heterotetramer)方有酵素活性；研究指出這些”異型”單元體中，其中一個單元體和一般已被廣泛研究之類異戊二烯轉移酵素一樣，都具有高度保留區域 (conserved motif)，另一個單元體則否。們研究的二異戊二烯焦磷酸合成酵素 (geranyl pyrophosphate synthase，GPPs)，在植物中可催化一個異戊二烯焦磷酸 (IPP)與二甲烯丙烯基焦磷酸 (dimethylallyl pyrophosphate，DMAPP)反應產生二異戊二烯焦磷酸 (geranyl pyrophosphate，GPP)。此產物是生物體內重要分子的前驅物，以植物而言，此產物能衍生出不同的氣味，在植物體中扮演著生殖及防禦功能；亦為植物骨架及二次代謝產物的前驅物。本論文中，首度解出薄荷之二異戊二烯焦磷酸合成酵素之結構。不同於以往已知結構的”同型”二聚體，此酵素之立體結構為”異型”四聚體，分別由兩個large單元體和兩個small單元體所組成。large單元體和一般已被廣泛研究之類異戊二烯轉移酵素在胺基酸序列比對中有高度相似性 (sequence homology);如同過去的研究，large單元體亦是利用環狀物 (loop)連接α螺旋 (α-helix)形成活性催化區，其催化中心藉由兩個DDXXD motif，在鎂離子的螯合下和受質 (substrates)反應。然而， small單元體的胺基酸序列卻沒有發現此相似性特徵；研究也發現small單元體能和不同物種之四異戊二烯焦磷酸合成酵素 (GGPPs)結合，限制其四異戊二烯焦磷酸合成酵素只能進行一次催化反應。據large單元體和芥菜 (Sinapis alba)四異戊二烯焦磷酸合成酵素在胺基酸序列 (74%)及結構上活性區域的受質結合區域的高度相似性，我們推測large單元體本身具四異戊二烯焦磷酸合成酵素的功能，即合成20碳產物的能力。進一步分析large和small單元體和其他此類酵素立體結構的差異，我們認為small單元體可能藉由其結構上的一個環狀物 (loop)，來調控large單元體的催化機制，限制large單元體只能進行一次催化反應，使其由原本產生20碳的產物改變為產生10碳的產物。藉由首度解出的”異型”類異戊二烯轉移酵素的結構，我們希望能對此種”異型”酵素結構的反應機制及其特異性有進一步的了解。Isoprenoids constitute perhaps the most diverse of natural products serving as vital functions in all living being. All isoprenoids are constructed from one or both of two basic precursors, C5-dimethylallyl pyrophosphate (DMAPP) and C5-isopentenyl pyrophosphate (IPP). Here, geranyl pyrophosphate synthase (GPPs), belonging to trans-prenyltransferases forming all-trans prenyl diphosphates through trans double-bond addition, catalyzes the condensation of C5- DMAPP with C5-IPP to generate C10-Geranyl pyrophosphate (GPP), a precursor of monoterpenes which function as defensive agents, pollination attractants, and major floral scent compounds in plant. Intriguingly, distinct from other solved structures of trans-prenyltransferases, which are homodimers or homohexamer, Mentha x piperita GPPs is proven to be as a functional heterotetramer, composing of two large subunits (2L) and small subunits (2S). The large subunit has the conserved functional motif, DDXXD, and a high amino acid sequence identity with geranylgeranyl pyrophosphate synthase (GGPPs). Notably, the small subunit has neither the conserved motif nor the sequence identify with other trans-prenyltransferases. In addition, previous studies indicated that the small subunit involved in product formation and substrate specificity and interacted with the large subunit and other GGPPs. Moreover, spatial and temporal expressions of small subunit in plant revealed closely correlating with monoterpene emission in contrast to large subunit having constitutively expressed in all developmental and flowering stages. Thus, the distinct roles of small and large subunit in the regulation mechanism and quaternary structure formation of heterotetramer (2L2S) in vitro and vivo still remain elusive. ere, we use structural biological and biochemical approaches to elucidate the structural properties of Mentha x piperita GPPs. At first, to improve the poor yield of recombinant GPPs expressed in Escherichia coli, both large and small subunit carrying the thioredoxin fusion tag in front of their N-terminus can significantly increase approximately 10-fold protein yield. But, due to the bottleneck of further purification and isolation, coexpression of small subunit with His-tag in its C-terminus and large subunit is applied to high-level purity and functional heterotetrameric proteins. The first heterotetrameric (2L2S) Mentha x piperita GPPs structures, including apo-form and complex with DMAsPP, IPP, and Mg2+ ions, were determined to resolutions of 1.9 &Aring; and 2.2 &Aring;, respectively. Large subunit is composed of 12α-helices joined by connecting loops around a central cavity containing two DDXXD motif at the top for substrate binding; Small subunit is composed of 11α-helices to form the similar structure fold to trans-prenyltransferases. By comparing the large subunit of Mentha x piperita GPPs with the solved structure of Sinapis alba GGPPs, it reveals that they have not only high amino acid sequence identity (about 74%) but also very similar structure, especially in their catalytic sites. In addition, based on functional assay, Mentha x piperita GPPs can generate C10-GPP and C20-GGPP when the sufficient substrates and enough catalytic reaction time are provided. Therefore, we suggest that large subunit may exhibit a function of GGPPs. Intriguingly, small subunit with the enzymatic activity of GPPs is observed here in contrast to the previous studies which mention no detectable prenyltransferase activity. Thus, why small subunit, a kind of GPPs, must interact with large subunit, functional GGPPs? The mutagenesis and enzyme kinetic assay will used to elucidate these mystery questions.TABLE OF CONTENTS文摘要 1BSTRACT 3BBREVIATIONS 5NTRODUCTION 7-1 Characteristics and Biosynthesis of Isoprenoids 7-2 Classification of Prenyltransferases 7-3 Isoprenyl Pyrophosphate Synthases (IPPSs) 8-4 Basic Catalytic Mechanism of trans-prenyltransferases 10-5 Monoterpene Biosynthesis in Plant 11-6 The Motive of This Study 12-7 Specific Aim of This Study 13ATERIALS AND METHODS 15-1 Chemicals and Reagents 15-2 Construction of Expression Vectors 15-3 Protein Expression 17-4 Protein Purification 18-4-1 Purification of Mature Proteins without Enterokinase Cutting Site 18-4-2 Purification of Mature Proteins with Enterokinase Cutting Site 19-4-3 Ion-exchange Chromatography 19-5 Crystallization and Data Collection 20-6 Structure Determination and Refinement 20-7 Western Blotting 21-8 Gel Filtration Chromatography 22-9 Mass Spectrometry Analysis 22-10 Final Products Formation and Analysis 22ESULTS 24-1 Analysis of Expression and Solubility of GPPs 24-1-1 Evaluation of Expression and Solubility Condition 24-1-2 Design, Expression, and Purification of GPPs Fusion Constructs 25-2 Purification of GPPs for Crystallization 26-3 Quaternary Structure of GPPs 26-4 Crystal Structure of Mentha x piperita GPPs 27-4-1 Overall Structure 27-4-2 Active Site Structure 28-4-3 Structure Comparison 28-5 Enzyme Activity of mint GPPs 30ISCUSSION 31ONCLUSION 34EFERENCES 35IGURES AND TABLES 4

Structure and Mechanism of an Arabidopsis Medium/Long-Chain-Length Prenyl Pyrophosphate Synthase1[W][OA]

Prenyltransferases (PTSs) are involved in the biosynthesis of terpenes with diverse functions. Here, a novel PTS from Arabidopsis (Arabidopsis thaliana) is identified as a trans-type polyprenyl pyrophosphate synthase (AtPPPS), which forms a trans-double bond during each homoallylic substrate condensation, rather than a homomeric C10-geranyl pyrophosphate synthase as originally proposed. Biochemical and genetic complementation analyses indicate that AtPPPS synthesizes C25 to C45 medium/long-chain products. Its close relationship to other long-chain PTSs is also uncovered by phylogenetic analysis. A mutant of contiguous surface polar residues was produced by replacing four charged surface amino acids with alanines to facilitate the crystallization of the enzyme. The crystal structures of AtPPPS determined here in apo and ligand-bound forms further reveal an active-site cavity sufficient to accommodate the medium/long-chain products. The two monomers in each dimer adopt different conformations at the entrance of the active site depending on the binding of substrates. Taken together, these results suggest that AtPPPS is endowed with a unique functionality among the known PTSs