抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

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Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

The present review offers an overview of nonclassical (e.g., with no pre- or in situ activation of a carboxylic acid partner) approaches for the construction of amide bonds. The review aims to comprehensively discuss relevant work, which was mainly done in the field in the last 20 years. Organization of the data follows a subdivision according to substrate classes: catalytic direct formation of amides from carboxylic and amines (section 2); the use of carboxylic acid surrogates (section 3); and the use of amine surrogates (section 4). The ligation strategies (NCL, Staudinger, KAHA, KATs, etc.) that could involve both carboxylic acid and amine surrogates are treated separately in section 5.

Nonclassical Routes for Amide Bond Formation

Protein farnesylation and geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming activity of many oncogenic proteins, including some RAS family members. This observation prompted the development of inhibitors of farnesyltransferase (FT) and geranylgeranyl-transferase 1 (GGT1) as potential anticancer drugs. In this Review, we discuss the mechanisms by which FT and GGT1 inhibitors (FTIs and GGTIs, respectively) affect signal transduction pathways, cell cycle progression, proliferation and cell survival. In contrast to their preclinical efficacy, only a small subset of patients responds to FTIs. Identifying tumours that depend on farnesylation for survival remains a challenge, and strategies to overcome this are discussed. One GGTI has recently entered the clinic, and the safety and efficacy of GGTIs await results from clinical trials.

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Targeting protein prenylation for cancer therapy

Lu Shin Wong graduated with a Bachelor of Pharmacy from the University of Nottingham in 1997. He then practiced as a hospital pharmacist for four years while completing his postgraduate diploma in clinical pharmacy from the University of Bradford. Subsequently, he undertook his Ph.D. studies at the University of Southampton with Prof. Mark Bradley on solid-phase organic chemistry, microspectrometry, and solid-supported sensors. Upon completing this in 2005, he joined the Manchester Interdisciplinary Biocentre as a postdoctoral research associate with Prof. Jason Micklefield on the application of chemical biology to surface chemistry and biomolecular-array technologies. Lu Shin is currently an EPSRC Life Science Interface research fellow, and his interests include the combination of chemical biology, surface chemistry, and nanofabrication towards life science applications.
Biography
Farid Khan obtained his Ph.D. from the University of Cambridge (2004), where he studied the folding of GFP using fluorescence and NMR. Previously, he has worked for a number of years at GlaxoSmithKline in fluorescence assay development for drug discovery. He spent two years as a postdoctoral researcher at the Babraham Institute in Cambridge, where he codeveloped novel protein arrays from DNA arrays using cell free synthesis and characterized robust protein immobilization methods. He is currently employed at the University of Manchester as a Systems Biologist at the Manchester Centre of Integrative Systems Biology. His primary role is on the characterizing of enzymes in metabolic pathways, and he is a M.Sc. lecturer in Biotechnology and Enterprise. He is a founder of Lumophore Ltd., a consulting company specializing in applications of protein array technology.
Biography
Jason Micklefield graduated from the University of Cambridge in 1993 with a Ph.D. in Organic Chemistry, working with Prof. Sir Alan R. Battersby to complete the first total synthesis of Haem d1. He then moved to the University of Washington, U.S.A., as a NATO postdoctoral fellow with Prof. Heinz G. Floss, investigating various biosynthetic pathways and enzyme mechanisms. In 1995 he began his independent research career as a Lecturer in Chemistry at Birkbeck College, University of London, before moving to Manchester in 1998, where he is now Chair of Chemical Biology. Prof. Micklefield?s research interests are at the chemistry?biology interface and include the redesign of nucleic acids, small-molecule control of gene expression, biosynthesis and biosynthetic engineering, nonribosomal peptides, biocatalysis, and enzyme mechanism.

Selective covalent protein immobilization: strategies and applications.

ATP-binding cassette (ABC) transporters comprise a superfamily of proteins, which actively transport a variety of compounds across cell membranes. Mammalian and most eukaryotic ABC transporters function as exporters, flipping or extruding substrates from the cytoplasmic to the extracellular or lumen side of cell membranes. Prokaryotic ABC transporters function either as exporters or importers. Here we show that ABCA4, an ABC transporter found in retinal photoreceptor cells and associated with Stargardt macular degeneration, is a novel importer that actively flips N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of disc membranes, thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptors. ABCA4 also actively transports phosphatidylethanolamine in the same direction. Mutations known to cause Stargardt disease decrease N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine transport activity of ABCA4. These studies provide the first direct evidence for a mammalian ABC transporter that functions as an importer and provide insight into mechanisms underlying substrate transport and the molecular basis of Stargardt disease.

/pdf/abca4-is-an-n-retinylidene-phosphatidylethanolamine-and-55s1krzxge.pdf

ABCA4 is an N -retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer

Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.

Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide (C10) or farnesylazide (C15) moiety from the corresponding prenyldiphosphates to a model peptide substrate, N-dansyl-Gly-Cys-Val-Ile-Ala-OH The rates of incorporation for these two substrate analogs are comparable and approximately twofold lower than that using the natural substrate farnesyl diphosphate (FPP) Reaction of N-dansyl-Gly-Cys(S-farnesylazide)-Val-Ile-Ala-OH with 2-diphenylphosphanylbenzoic acid methyl ester then gives a stable alkoxy-imidate linked product This result suggests future generations whereby azide groups introduced using this enzymatic approach are functionalized using a broad range of azide-reactive reagents Thus, chemistry has been developed that could be used to achieve highly specific peptide and protein modification The farnesylazide analog may be useful in certain biological studies, whereas the geranylazide group may be more useful for general protein modification and immobilization

Evaluation of geranylazide and farnesylazide diphosphate for incorporation of prenylazides into a CAAX box-containing peptide using protein farnesyltransferase.

Selective labeling of polypeptides using protein farnesyltransferase via rapid oxime ligation

To obtain a transition state (TS) structure for an enzyme-catalyzed prenylation reaction, SN1 and SN2 model substitution reactions with dimethylallyl chloride were first studied. 13C Kinetic isotope effects (KIEs) for the model reactions were measured by a natural abundance NMR method and used to validate the computational methods that would be used in the subsequent determination of the enzymatic TS structure. Using a primary 13C KIE and a secondary 2H KIE measured via mass spectrometry, a TS structure for the enzyme-catalyzed reaction was computed; a density functional level of electronic structure theory using the mPW1N functional in combination with the 6-31+G(d) basis set was employed for those calculations. That structure has a C−O bond length of 1.69 A and a C−S bond length of 3.70 A. While the former bond length is similar to that for a nonenzymatic SN2 reaction, the latter is considerably (0.90 A) longer, indicating that the enzyme effects catalysis via an “exploded” TS structure.

Stepan Lenevich

Papers

ABCA4 is an N -retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer

Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation

Evaluation of geranylazide and farnesylazide diphosphate for incorporation of prenylazides into a CAAX box-containing peptide using protein farnesyltransferase.

Selective labeling of polypeptides using protein farnesyltransferase via rapid oxime ligation

Transition state analysis of model and enzymatic prenylation reactions