2009

Ionic interactions promote transmembrane helix-helix association depending on sequence context

J. Mol. Biol., 2009, published on 01.12.2009
Journal of Molecular Biology, online article
Folding and oligomerization of integral membrane proteins frequently depend on specific interactions of transmembrane helices. Interacting amino acids of helix-helix interfaces may form complex motifs and exert different types of molecular forces. Here, a set of strongly self-interacting transmembrane domains (TMDs), as isolated from a combinatorial library, was found to contain basic and acidic residues, in combination with polar nonionizable amino acids and C-terminal GxxxG motifs. Mutational analyses of selected sequences and reconstruction of high-affinity interfaces confirmed the cooperation of these residues in homotypic interactions. Probing heterotypic interaction indicated the presence of interhelical charge-charge interactions. Furthermore, simple motifs of an ionizable residue and GxxxG are significantly overrepresented in natural TMDs, and a specific combination of these motifs exhibits high-affinity heterotypic interaction. We conclude that intramembrane charge-charge interactions depend on sequence context. Moreover, they appear important for homotypic and heterotypic interactions of numerous natural TMDs

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Phosducin influences sympathetic activity and prevents stress-induced hypertension in humans and mice

Journal of Clinical Investigation, 2009, 119 (12), 3597 - 3612 published on 23.11.2009
The Journal of Clinical Investigation, online article
Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individual’s lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc–/– mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc–/– mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc–/– mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene–based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12–15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.

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Synthesis of Threefold Glycosylated Proteins using Click Chemistry and Genetically Encoded Unnatural Amino Acids

ChemBioChem, 2009, 10, 18, 2858 - 2861 published on 06.11.2009
ChemBioChem, online article
Eukaryotic proteins and in particular cell-surface proteins are frequently glycosylated, which has fueled the interest of chemist to develop new methods for the synthesis of glycosylated proteins. Protein glycosylation is essential for the proper function of the respective proteins and in the case of glycosylated protein therapeutics, such as erythropoietin, the activity of the protein strongly depends on glycosylation patterns. The synthesis and production of glycosylated proteins to either elucidate their biochemical function or to bring a new therapeutic to the market is a formidable challenge. The current most widely used methods for the production of glycosylated proteins involve over-expression of the protein in particular host systems, such as mammalian or plant cell cultures, moss cultures or tobacco plants, to name a few, that are able to make either partial or even fully glycosylated proteins. Alternatively, solid-phase synthesis of glycosylated peptides and their insertion into the protein in question by native or expressed chemical ligation has emerged as a powerful chemical strategy.

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Light Commands Ion Channel

Chemical & Engineering News, 2009, published on 04.11.2009
Online article
Chemical Biology: Photosensitive reaction opens or shuts potassium's flow. Drugs that are modulated by light may have a more controllable cousin on the horizon. Researchers led by Dirk Trauner, a chemist at the University of Munich, have figured out the mechanism by which molecules with the ability to block voltage-gated ion channels can be turned on and off repeatedly by light. Because ion channels are at the foundation of human perception, these light-sensitive ion channel blockers, also called photochromic ligands, could one day be used in light-controlled anesthesia or vision restoration, Trauner says.

Light has long been used to activate drugs, leading to several successful so-called photodynamic therapies, such as those for treating cancer. More recently, Trauner and his colleagues have spearheaded an effort to turn on molecules that target biological proteins—and then turn them off again—by means of light-activated cis-trans isomerization, an emerging field they call photopharmacology.

Early photochromic ligands could bind only to ion channels that had been genetically modified to permit attachment. Last year, Trauner and colleagues reported an azobenzene molecule dubbed AAQ that can photosensitize naturally occurring voltage-gated potassium channels in neurons (Nature Methods 2008, 5, 331). Now the team is reporting AAQ’s mechanism of action, along with a whole family of related azobenzenes that can block voltage-gated potassium ion channels in a photo-controllable manner (Angew. Chem. Int. Ed., 10.1002/ange.200904504). In particular, the team finds that AAQ diffuses into brain cells and binds noncovalently in an area of the channel interior in which potassium ions would normally pass. In the trans configuration, AAQ blocks the channel, whereas in the cis configuration, it allows potassium ions to flow.

Given the similar structure, function, and pharmacology of voltage-gated ion channels, Trauner's "beautiful" new study points to the possibility of creating photochromic ligands for other such channels, such as voltage-gated calcium channels, comments Stefan Herlitze, a neuroscientist at Case Western Reserve University. One could imagine that specific light-gated blockers could trigger the firing of neurons, which could also be important in heart disease, he adds.

But unless the molecules are made selective for specific channels in specific cell types, their therapeutic potential will be limited, say other researchers, including biochemist Timothy Ryan at Cornell University.

"We are also concerned with selectivity," Trauner says. In as-yet-unpublished work, his group has found that the molecules can be made more selective by chemically tweaking their hydrophobic tails.

 

 

 

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Microsolvated and Chelated Butylzinc Cations: Formation, Relative Stability, and Unimolecular Gas-Phase Chemistry

Chem. Eur. J., 2009, 15(46), 12745-12753, doi 10.1002/chem.200901963 published on 23.10.2009
Chem. Eur. J., online article
Solutions of butylzinc iodide in tetrahydrofuran, acetonitrile, and N,N-dimethylformamide were analyzed by electrospray ionization mass spectrometry. In all cases, microsolvated butylzinc cations [ZnBu(solvent)n]+, n=1-3, were detected. The parallel observation of the butylzincate anion [ZnBuI2]- suggests that these ions result from disproportionation of neutral butylzinc iodide in solution. In the presence of simple bidentate ligands (1,2-dimethoxyethane, N,N-dimethyl-2-methoxyethylamine, and N,N,N',N'-tetramethylethylenediamine), chelate complexes of the type [ZnBu(ligand)]+ form quite readily. The relative stabilities of these complexes were probed by competition experiments and analysis of their unimolecular gas-phase reactivity. Fragmentation of mass-selected [ZnBu(ligand)]+ leads to the elimination of butene and formation of [ZnH(ligand)]+. In marked contrast, the microsolvated cations [ZnBu(solvent)n]+ lose the attached solvent molecules upon gas-phase fragmentation to produce bare [ZnBu]+, which subsequently dissociates into [C4H9]+ and Zn. This difference in reactivity resembles the situation in organozinc solution chemistry, in which chelating ligands are needed to activate dialkylzinc compounds for the nucleophilic addition to aldehydes.

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Combination of FASP and StageTip-Based Fractionation Allows In-Depth Analysis of the Hippocampal Membrane Proteome

Journal of Proteom Research, 2009, 8, 12, 5674 - 78 published on 22.10.2009
Journal of Proteome Research, online article
Membrane proteomics is challenging because the desirable strong detergents are incompatible with downstream analysis. Recently, we demonstrated efficient removal of SDS by the filter aided sample preparation method (FASP). Here we combine FASP with our previously described small-scale membrane enrichment protocol. Analysis of a single mouse hippocampus enables identification of more than 1000 membrane proteins in a single LC-MS/MS run without protein or peptide prefractionation. To extend proteome coverage, we developed a simple anion exchange fractionation method in a StageTip format. When separating peptides into six fractions, a duplicate analysis resulted in identification of 4206 proteins of which 64% were membrane proteins. This data set covers 83% of glutamate and GABA receptor subunits identified in hippocampus in the Allen Brain Atlas and adds further isoforms. The combined method provides a streamlined protocol for rapid and sensitive membrane proteome mapping. We also provide a generic protocol for combining FASP with StageTip-based ion exchange fractionation, which is generally applicable to proteome analysis.

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CIPSM PI and board member Arne Skerra and his new spin-off company XL-protein GmbH prolong the plasma half-life of biopharmaceutical substances

2009, published on 19.10.2009

http://www.xl-protein.com

Many biopharmaceuticals comprise small proteins that are quickly eliminated from the body. CIPSM PI and board member Arne Skerra and his new spin-off XL-protein GmbH combine such small proteins with a kind of molecular balloon that swells and thus prolongs the half-life of these proteins in the body. The CIPSM spin-off XL-protein GmbH has now started to further develop this new technology with blockbuster potential. People who suffer from hepatitis B are often treated with the tissue hormone interferon. However, there is a problem: Interferon is a very small protein, which is filtered from the blood via the kidneys after only a short time. For the patient this means a high-dose injection every other day to keep the effect of the substance from wearing off prematurely. However, interferon stays in the body much longer when chemically coupled with a synthetic PEG (polyethylene glycol) molecule. PEG is a random coil long-chain polymer string that swells by adsorbing water. That way the PEG molecule becomes large enough that it does not fit through the fine pores of the kidneys – the attached interferon remains in the circulatory system longer, and the patient will need an injection only every one to two weeks. Using genetic engineering, CIPSM and TUM scientist Prof. Arne Skerra and his coworkers from the Chair of Biological Chemistry at the Center for Life and Food Sciences Weihenstephan have now developed an amino acid string that tangles up similarly to PEG and also swells in the presence of water. However, unlike many PEG compounds, there is no danger of this biological polymer accumulating in the body. In fact – over an extended period of time – it is discharged or biologically broken down. That happens because this amino acid string (polypeptide) consists of three of the 20 naturally occurring amino acids: proline, alanine and serine, or in short, PAS. The protein substance interferon, which itself consists of amino acids, can thus be easily generated in "PASylated" form. In first trials with animals, CIPSM scientists established that PASyated interferon has a half-life in the blood that is prolonged by a factor of 60, which should allow a significant extension of dosing intervals during medicinal therapy. A further advantage is the simplified biotechnological production: The DNA segments carrying the information for the PAS amino acid sequence and for the interferon can simply be attached to each other and then, for instance, used for transforming bacteria. The bacteria then produce the PASylated interferon in one piece, thus making much fewer production steps necessary in comparison with the chemical coupling of PEG. According to Skerra, "this will lead to a significant drop in production cost." In principle all small proteins currently used as medication or in development in pharmaceutical companies – for example, growth factors or functional antibody fragments – can be PASylated. Thus there could be a huge market for the new technology. Consequently, Prof. Skerra and his team initiated the founding of a new biotech company, XL-protein GmbH (http://www.xl-protein.com), which started its operations last spring. "Our technology has the potential to give birth to a whole new generation of blockbuster medications," the Arne Skerra is convinced. Several of the new drugs are already at an advanced stage of preclinical development.

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DynaDock: A new molecular dynamics-based algorithm for protein-peptide docking including receptor flexibility

Proteins: Structure, Function, and Bioinformatics, 2010, 78(5), 1084 - 1104, doi:10.1002/prot.22629 published on 16.10.2009
Proteins, online article
Molecular docking programs play an important role in drug development and many well-established methods exist. However, there are two situations for which the performance of most approaches is still not satisfactory, namely inclusion of receptor flexibility and docking of large, flexible ligands like peptides. In this publication a new approach is presented for docking peptides into flexible receptors. For this purpose a two step procedure was developed: first, the protein-peptide conformational space is scanned and approximate ligand poses are identified and second, the identified ligand poses are refined by a new molecular dynamics-based method, optimized potential molecular dynamics (OPMD). The OPMD approach uses soft-core potentials for the protein-peptide interactions and applies a new optimization scheme to the soft-core potential. Comparison with refinement results obtained by conventional molecular dynamics and a soft-core scaling approach shows significant improvements in the sampling capability for the OPMD method. Thus, the number of starting poses needed for successful refinement is much lower than for the other methods. The algorithm was evaluated on 15 protein-peptide complexes with 2-16mer peptides. Docking poses with peptide RMSD values <2.10 Å from the equilibrated experimental structures were obtained in all cases. For four systems docking into the unbound receptor structures was performed, leading to peptide RMSD values <2.12 Å. Using a specifically fitted scoring function in 11 of 15 cases the best scoring poses featured a peptide RMSD 2.10 Å.

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A Photolabile Linker for the Mild and Selective Cleavage of Enriched Biomolecules from Solid Support

JOC, 2009, 74 Issue 21, 8476–9 published on 12.10.2009
JOC, online article
Selective release of enriched biomolecules from solid support is a desirable goal in proteomic and metabolomic studies. Here we demonstrate that photocleavage of a light-sensitive phenacyl ester bond is a suitable alternative cleavage strategy for the selective release of enriched biomolecules form avidin beads circumventing the disadvantages of conventional heat denaturation procedures.

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A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity

Acta Chrystallogr. D, 2009, 65(10), 1118 - 1125, doi:10.1107/S0907444909031011 published on 01.10.2009
Acta Chrystallographica D, online article
Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P21 with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6 Å resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P21 crystal form uncovered major conformational changes (i) in β-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the β-barrel and (iii) in the extended C-terminal segment, which is attached to the β-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family.

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Parallel Isotope-Based Quantification of Modified tRNA Nucleosides

Angew. Chem. Int. Ed., 2009, 48, 1–4, DOI:10.1002/anie.200902740 published on 11.09.2009
Angew. Chem. Int. Ed., online article
Modifications make a difference: An isotope-based mass spectrometry method allows the facile and quantitative analysis of modified tRNA nucleosides in various types of cells. This method could be capable of distinguishing between individual cell lines as well as between healthy tissue and cancer cells.

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Syringolin A Selectively Labels the 20S Proteasome in Murine EL4 and Wild-Type and Bortezomib-Adapted Leukaemic Cell Lines

ChemBioChem, 2009, DOI: 10.1002/cbic.200900411 published on 11.09.2009
ChemBioChem, online article
The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

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Once Overlooked, Now Made Visible: ATL Proteins and DNA Repair

Angew. Chem. Int. Ed., 2009, 48, 2–5, DOI:10.1002/anie.200904042 published on 08.09.2009
Angew. Chem. Int. Ed., online article
The DNA of a cell is continuously exposed to numerous endogenous and exogenous factors. The resulting DNA damage can lead to mutations or cell death. Some of the major DNA lesions are generated by the reaction of alkylating reagents with DNAbases. The alkylated reaction products can arise endogenously from cellular alkylating reagents as Sadenosylmethionine or from the influence of exogenous factors (e.g. environmental stress). In cancer therapy, alkylating agents are also used to damage the DNA of tumor cells, resulting in various alkylated bases. In addition to the N7 position of guanine and adenine, the O6 position of guanine and the O4 position of thymine are susceptible to alkylation. It is known that O6-methylguanine pairs with thymine during replication, resulting in a G·C to A·T transition mutation.

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Crystal Structure of the T(6-4)C Lesion in Complex with a (6-4) DNA Photolyase and Repair of UV-Induced (6-4) and Dewar Photolesions

Chem. Eur. J., 2009, DOI: 10.1002/chem.200901004, published on 31.08.2009
Chem. Eur. J., online article
UV-light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis-syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6-4)pyrimidone photoproducts ((6-4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6-4)C lesion containing DNA duplex in complex with the (6-4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6-4) lesions into strained four-membered ring repair intermediates, it is surprising that the not rearranged T(6-4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6-4) DNA photolyases

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Snapshots of the Fluorosalinosporamide/20S Complex Offer Mechanistic Insights for Fine Tuning Proteasome Inhibition

Journal of Medicinal Chemistry, 2009, 52, 17, 5420 - 28 published on 13.08.2009
Journal of Medicinal Chemistry, online article
Many marketed drugs contain fluorine, reflecting its ability to modulate a variety of biological responses. The unique 20S proteasome inhibition profile of fluorosalinosporamide compared to chlorinated anticancer agent salinosporamide A (NPI-0052) is exemplary and relates to each halogen’s leaving group potential. Crystal structures of fluoro-, hydroxy-, and bromosalinosporamide in complex with the yeast 20S proteasome core particle (CP) provide mechanistic insights into ligand binding and leaving group elimination and the ability to fine-tune the duration of proteasome inhibition. Fluorosalinosporamide/CP crystal structures determined over time offer striking snapshots of the ligand trapped with an intact fluoroethyl group in anticipation of fluoride elimination, followed by complete nucleophilic displacement of fluoride to give the highly stabilized cyclic ether found for salinosporamide A and bromosalinosporamide. This two-step reaction pathway is consistent with a mechanism for partially reversible proteasome inhibition by fluorosalinosporamide. Proteasome catalyzed fluoride displacement provides preliminary insights into the active site Thr1N pKa.

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beta-Lactones Decrease the Intracellular Virulence of Listeria monocytogenes in

ChemMedChem, 2009, 8, 1260-3 published on 04.08.2009
ChemMedChem, online article
The increasing emergence of multiresistant bacterial pathogens represents a dramatic global health problem. One major reason for this dilemma is the high selective pressure that is exerted on bacteria by classical antibiotic therapies, leading to a steady increase in the number of strains resistant to the majority of all currently available antibiotic drugs. In addition, only a limited number of essential molecular targets in bacteria such as cell wall and protein biosynthesis have been systematically exploited by a small number of antibiotic scaffolds. It is therefore a major goal to identify novel targets and corresponding drugs that are either important for bacterial viability or essential for their pathogenicity and virulence.[1, 2] Inhibition of virulence-associated targets would not necessarily kill bacteria, but disarm their pathogenic weapon repertoire. This could then lead to the elimination of disarmed bacteria by the host immune response and could additionally exert less selective pressure.[3, 4]

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Prolonged stability by cyclization: Macrocyclic phosphino dipeptide isostere inhibitors of ß-secretase (BACE1)

Bioorg. Med. Chem. Lett., 2009, 19(15), 4427-4431, doi:10.1016/j.bmcl.2009.05.053 published on 01.08.2009
Bioorganic & Medicinal Chemistry Letters, online article
Cyclization of recently reported linear phosphino dipeptide isostere inhibitors of BACE1 via side chain olefin metathesis yielded macrocyclic BACE1 inhibitors. The most potent compound II-P1 (IC50 of 47 nM) and the corresponding linear analog I were tested for serum stability. The approach led to three times prolonged half life serum stability of 44 min for the macrocyclic inhibitor II-P1 compared to the linear compound I.

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A new ground state single electron donor for excess electron transfer studies in DNA

Chem. Comm., 2009, 24, 3583-3584 published on 28.07.2009
Chem. Comm., online article
A new photoinducible single electron donor has been developed, which, when linked to thymidine, is shown to be an efficient ground state reducing agent in DNA; the donor can be activated at wavelengths where standard DNA does not absorb.

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Structure of Active IspH Enzyme from Escherichia coli Provides Mechanistic Insights into Substrate Reduction

Angewandte Chemie, 2009, Doi: 10.1002/anie.200900548, published on 30.06.2009
Angewandte Chemie, online article
Eukaryotes and most prokaryotes require isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as biosynthetic precursors of terpenes. Whereas animals generate these essential metabolites via the mevalonate pathway,[1] many human pathogens including Plasmodium falciparum and Mycobacterium tuberculosis are known to use the more recently identified non-mevalonate pathway, which is a potential target for drug development.[2–4] The final step of this pathway is catalyzed by IspH protein, which generates a mixture of IPP and DMAPP by reductive dehydration of 1- hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP, Figure 1a).[5–11] Recently, Rekittke et al. described the first X-ray structure of IspH protein from the hyperthermophilic eubacterium Aquifex aeolicus in its open state.[12] Herein, we report the crystal structure of the IspH protein from Escherichia coli[11] in its closed conformation, which serves as basis for a detailed discussion of the catalytic pathway

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NMR Structure and Dynamics of the Engineered Fluorescein-Binding Lipocalin FluA Reveal Rigidification of β-Barrel and Variable Loops upon Enthalpy-Driven Ligand Binding

Biochem., 2009, 48 (31), 7411–7419, doi:10.1021/bi900535j published on 29.06.2009
Biochemistry, online article
The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel β-strands forming a β-barrel with an α-helix attached to its side. Comparison of the NMR structure of free FluA with the X-ray structures of BBP·biliverdin IXγ and FluA·fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the β-barrel as well as adjoining β-strand segments. An “induced fit” became apparent for the side chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse 15N backbone spin relaxation times in the rotating frame for free FluA and also for the FluA·fluorescein complex. A reduction in the level of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy-driven, thus overcompensating for the negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin−ligand interaction not only provides insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but also supports the future design of corresponding binding proteins with novel specificities, so-called “anticalins”.

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Alpenforum 2009

published on 19.06.2009
CIPSM Events
CIPSM sponsors the "Alpenforum 2009" which will rock Oberammergau from 26.-28. of June 2009! The event is dedicated to straighten contacts between young chemists and companies. Be there or be square!

 

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Structural biology of DNA photolyases and cryptochromes

Curr. Opin. Struct. Biol., 2009, 19, 277-285 published on 30.05.2009
Curr. Opin. Struct. Biol., online article
Photolyases repair cytotoxic and mutagenic UV-induced photolesions in DNA by using an amazing light-dependent repair mechanism. It involves light absorption, electron transfer from an excited reduced and deprotonated FADH− to the flipped-out photolesion, followed by the fragmentation of the photolesions. Cryptochromes are highly related proteins that no longer repair damaged DNA, but function as photoreceptors. They feature strikingly similar protein architectures to photolyases and contain an FAD cofactor as well. However, cryptochromes possess an additional signal-transmitting domain, attached either to the N-termini or C-termini. Recently, the field of photorepair and blue-light photoperception has experienced significant progress particularly in structural biology, which is summarized in this review. Today, crystal structures of many family members are known and most recently even complexes of photolyases and DASH-type cryptochrome bound to their DNA substrates became available providing insight into the critical electron and energy transfer reactions that enable genome repair.

CurrOpinStructBiol_2009_19_277

 

 

 



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CIPSM-Harvard Young Scientists' Forum (YSF)

published on 29.05.2009
CIPSM Events
The LMU-Harvard Young Scientists’ Forum (YSF) seeks to unite Ph.D. students and postdoctoral fellows from the Harvard University and the Ludwig-Maximilians-Universität (LMU) with core faculty from the two universities to create a framework for an interdisciplinary exchange of ideas. This year’s conference entiteled "From Molecules to Organisms" will be held at the Center for Advanced Studies of LMU Munich from June 21-24.

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Cinnamic aldehyde derived probes for the active site labelling of pathogenesis associated enzymes

Chemical Communications, 2009, DOI: 10.1039/b905527d, 3741- 43 published on 19.05.2009
Chemical Communcations, online article
Michael acceptor based natural product derived probes are selective and sensitive chemical tools for the identification and characterization of pathologically relevant enzymes in MRSA.

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Interaction and conformational dynamics of membrane-spanning protein helices

Protein Science, 2009, 18(7), 1343-1358, doi:10.1002/pro.154 published on 13.05.2009
Protein Science, online article
Within 1 or 2 decades, the reputation of membrane-spanning -helices has changed dramatically. Once mostly regarded as dull membrane anchors, transmembrane domains are now recognized as major instigators of protein-protein interaction. These interactions may be of exquisite specificity in mediating assembly of stable membrane protein complexes from cognate subunits. Further, they can be reversible and regulatable by external factors to allow for dynamic changes of protein conformation in biological function. Finally, these helices are increasingly regarded as dynamic domains. These domains can move relative to each other in different functional protein conformations. In addition, small-scale backbone fluctuations may affect their function and their impact on surrounding lipid shells. Elucidating the ways by which these intricate structural features are encoded by the amino acid sequences will be a fascinating subject of research for years to come.

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Stabilization of conformationally dynamic helices by covalently attached acyl chains

Protein Science, 2009, 18(9), 1801-1805, doi: 10.1002/pro.155 published on 13.05.2009
Protein Science, online article
Acylation of proteins is known to mediate membrane attachment and to influence subcellular sorting. Here, we report that acylation can stabilize secondary structure. Circular dichroism spectroscopy showed that N-terminal attachment of acyl chains decreases the ability of an intrinsically flexible hydrophobic model peptide to refold from an -helical state to -sheet in response to changing solvent conditions. Acylation also stabilized the membrane-embedded -helix. This increase of global helix stability did not result from decreased local conformational dynamics of the helix backbone as assessed by deuterium/hydrogen-exchange experiments. We concluded that acylation can stabilize the structure of intrinsically dynamic helices and may thus prevent misfolding.

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Bilder von der CIPSM-Festveranstaltung im Deutschen Museum am 4. Mai 2009

published on 07.05.2009
CIPSM Events
Click here and keep on scrolling if you care for pictures form the event "CIPSM - Ausgewählter Ort im Land der Ideen 2009" which was held on the 4th of May 2009 in the Deutsche Museum.

 

Click here for all pictures

 

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An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies

PNAS, 2009, DOI: 10.1073/pnas.0813399106 published on 05.05.2009
PNAS, online article
Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this “antigen.” Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small (≈180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.

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CIPSM-Festveranstaltung im Deutschen Museum am 4. Mai 2009

published on 04.05.2009
CIPSM Events
CIPSM ist „Ausgewählter Ort im Land der Ideen“. Damit ist CIPSM Teil der Veranstaltungsreihe „365 Orte im Land der Ideen“, die gemeinsam von der Standortinitiative „Deutschland – Land der Ideen“ und der Deutschen Bank durchgeführt wird. Als „Ausgewählter Ort“ wird CIPSM unter der Schirmherrschaft von Bundespräsident Horst Köhler im Jahr 2009 Deutschland als das „Land der Ideen“ repräsentieren und die Innovationsfreude unseres Landes erlebbar machen. Aus Anlass der Preisverleihung beehrt sich CIPSM zu Empfang und Vorträgen zum Thema "Proteinforschung für wirksamere Medikamente" am Montag, 4. Mai 2009, 09:30 Uhr in den Ehrensaal des Deutschen Museums einzuladen.

 

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The archaeal cofactor F0 is a light-harvesting antenna chromophore in eukaryotes

PNAS, 2009, 106, 11504-11545 published on 01.05.2009
PNAS, online article
Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin (F0) and F420 are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle F420/F420H2. In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds F0. Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of F0 in complex with the D. melanogaster protein shows the atomic details of F0 binding and activation, allowing characterization of the residues involved in F0 activation. The results show that the F0/F420 coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the F0 cofactor also in higher eukaryotes.

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Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein

PNAS, 2009, DOI: 10.1073/pnas.0902688106 published on 28.04.2009
PNAS, online article
Prion disease is characterized by the α→β structural conversion of the cellular prion protein (PrPC) into the misfolded and aggregated “scrapie” (PrPSc) isoform. It has been speculated that methionine (Met) oxidation in PrPC may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrPC [rhPrPC(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrPC variant is an α-helix rich protein (like Met-rhPrPC) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrPC variant is a β-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrPC, the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the α→β secondary structure conversion in rhPrPC with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation.

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Natural product inhibitors of protein–protein interactions mediated by Src-family SH2 domains

Bioorganic & Medical Chemistry Letters, 2009, doi:10.1016/j.bmcl.2009.04.083, published on 23.04.2009
Bioorganic & Medical Chemistry Letters, online article
In this Letter, we report the natural products salvianolic acid A, salvianolic acid B, and caftaric acid as inhibitors of the protein–protein interactions mediated by the SH2 domains of the Src-family kinases Src and Lck, two established disease targets. Moreover, we propose a binding mode for the inhibitors based on molecular modeling, which will facilitate chemical optimization efforts of these important lead structures for drug discovery.

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Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

PNAS, 2009, 106/16, 6507 - 12 published on 21.04.2009
PNAS, online article
Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactin-based proteasome inhibitor described so far. With a Ki′ of 8.65 ± 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics.

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Universal sample preparation method for proteome analysis

Nature Methods, 2009, 6, 359 - 62 published on 19.04.2009
Nature Methods, online article
We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.

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Sequence-specific conformational dynamics of model transmembrane domains determines their membrane fusogenic function

Journal of Molecular Biology, 2009, 386, 733-41 published on 14.04.2009
Journal of Molecular Biology, online article
The transmembrane domains of fusion proteins are known to be functionally important and display an overabundance of helix-destabilizing Ile and Val residues. In an effort to systematically study the relationship of fusogenicity and helix stability, we had previously designed LV-peptides, a low-complexity model system whose hydrophobic core consists of Leu and Val residues at different ratios. The ability of LV-peptides to fuse membranes increases with the content of helix-destabilizing residues. Here, we monitored the kinetics of amide deuterium/hydrogen-exchange of LV-peptide helices to probe their conformational dynamics. The kinetics indeed increase strongly with the content of helix-destabilizing residues and are likely to reflect local fluctuations of the helix backbones as all peptides exhibit uncorrelated exchange and contain sub-populations of amide deuteriums that exchange with different velocities. Interestingly, helices whose amide deuteriums are shifted from slower into faster sub-populations are more fusogenic. To map functionally relevant helix subdomains, novel peptide variants were designed, where Val residues are concentrated at peripheral or central domains of the hydrophobic core, respectively. Their structural and functional analysis suggests that dynamic domains close to the helix termini are more relevant for fusogenicity than central domains but cooperate with the latter to achieve strong fusogenicity. We expect that studies provide new vistas for investigating the impact of transmembrane domains on lipid membranes.

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beta-Lactam Probes As Selective Chemical-Proteomic Tools for the Identification and Functional Characterization of Resistance Associated Enzymes in MRSA

Journal of the American Chemical Society, 2009, DOI: 10.1021/ja901304n, published on 08.04.2009
JACS, online article
With the development of antibiotic resistant bacterial strains, infectious diseases have become again a life threatening problem. One of the reasons for this dilemma is the limited number and breadth of current therapeutic targets for which several resistance strategies have evolved over time. To identify resistance associated targets and to understand their function, activity, and regulation, we utilized a novel strategy based on small synthetic β-lactam molecules that were applied in activity based protein profiling experiments (ABPP) to comparatively profile in situ enzyme activities in antibiotic sensitive and resistant S. aureus strains (MRSA). Several enzyme activities which are unique to the MRSA strain including known resistant associated targets, involved in cell wall biosynthesis and antibiotic sensing, could be identified. In addition, we also identified uncharacterized enzymes which turned out to be capable of hydrolyzing β-lactam antibiotics. This technology could therefore represent a valuable tool to monitor the activity and function of other yet unexplored resistance associated enzymes in pathogenic bacteria and help to discover new drug targets for customized therapeutic interventions.

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Copper-Free “Click” Modification of DNA via Nitrile Oxide-Norbornene 1,3-Dipolar Cycloaddition

Org. Lett., 2009, 11, 2405-2408 published on 01.04.2009
Org. Lett., online article
Nitrile oxides react smoothly and rapidly with norbornene-modified DNA in a copper-free click reaction. The reaction allows high density functionalization of oligodeoxyribonucleotides (ODNs) with a large variety of molecules directly on solid supports and even in synthesizers without the need for an additional catalyst.

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Protein Iodination by Click Chemistry

ChemBioChem, 2009, 10.1002/cbic.200800816, published on 31.03.2009
ChemBioChem, online article
Upon development of radioimmunoassay techniques,[1] radioiodination of peptides and proteins has gradually become an indispensable tool for controlling and monitoring protein and peptide functions in vivo and in vitro, and for studying ligand–receptor interactions, ligand uptake and clearance. In addition, protein iodination has developed into a useful technique for heavy-metal modification in crystallographic analysis,[2] and serves as a valuable alternative to the expression of proteins with chalcogen amino acid analogues, such as selenomethionine, telluromethionine and selenocysteine.[3] Direct iodination of peptides and proteins at histidine, and more efficiently at tyrosine residues, under the various oxidative conditions is accompanied by modification of sensitive amino acid residues, such as methionine, cysteine and particularly tryptophan.[4] Indirect iodination methods that are based mainly on conjugation of peptides and folded proteins at surface exposed amine functions with iodinated N-hydroxysuccinimidyl-3-(4-hydroxyphenyl) propionate (Bolton–Hunter reagent[5]), and more recently with N-succinimidyl 3-iodobenzoate, have, therefore, been proposed.

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CIPSM Movie online stream

CIPSM Movie, 23. March 2009, published on 23.03.2009
DFG comment box
2009 is great! If you have time, take 5 minutes to watch CIPSM on silverscreen.

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High-Affinity Recognition of Lanthanide(III) Chelate Complexes by a Reprogrammed Human Lipocalin 2

JACS, 2009, 131 Vol. 10, 3565-76 published on 18.03.2009
Human lipocalin 2 (Lcn2), also known as neutrophil gelatinase-associated lipocalin (NGAL), which naturally scavenges bacterial ferric siderophores, has been engineered to specifically bind rare-earth and related metal ions as chelate complexes with [(R)-2-amino-3-(4-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diaminepentaacetic acid (p-NH(2)-Bn-CHX-A''-DTPA). To this end, 12 amino acid residues in the ligand pocket of Lcn2, which is formed by four loops at the open end of an eight-stranded beta-barrel, were subjected to targeted random mutagenesis, and from the resulting library, variants with binding activity for the Me x DTPA group were selected using the method of bacterial phage display. One promising candidate was further developed in several cycles of in vitro affinity maturation using partial random mutagenesis and selection (via phage display and/or Escherichia coli colony screening) under conditions of increasing stringency. As result, an Lcn2 variant was obtained that binds Y x DTPA with a dissociation constant as low as 400 pM. The Lcn2 variant specifically recognizes the artificial ligand, as exemplified in (competitive) ELISA and real-time surface plasmon resonance analyses. DTPA-complexed Y(3+), Tb(3+), Gd(3+), and Lu(3+) are most tightly bound, comprising metal ions whose isotopes are in common use for radiotherapy and imaging. All of the Lcn2 variants are stably folded and can be functionally produced in high yield in E. coli. X-ray crystallographic analyses show that the new ligand is well-accommodated in the central cavity of the engineered lipocalin, whose fold is largely preserved, but that the mode of binding differs from the one seen with the natural ligand Fe x enterobactin. This structural study reveals analogies but also differences with respect to previously described antibody-metal chelate complexes. Notably, the functionalized side chain of DTPA protrudes from the ligand pocket of the lipocalin in such a way that its conjugates (with other haptens, for example) are recognized too. With their small sizes and robust fold based on a single polypeptide chain, the engineered Lcn2 variants provide novel modules and/or fusion partners for radionuclide-chelate capturing strategies that bear promise for medical diagnostics and therapy.

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Allosteric Switches: Remote Controls for Proteins

Angewandte Chemie International Edition, 2009, 48, 2 - 5 published on 11.03.2009
Angewandte Chemie, online article
Chemical biology has multiple aims,[1] one of which is the identification of small-molecule modulators for individual functions of as many human proteins as possible. Various approaches towards this goal have been developed, which converge to create a toolbox of small molecules for chemical biologists.[2–4] This endeavor constitutes a long and winding road, however, on which shortcuts are welcome. One intriguing shortcut would be to control the activity of any protein of interest by fusing it to a universally applicable “switch” protein, for which an experimentally accessible control mechanism exists (Scheme 1).

berg_angew_09_500



Unraveling the Secrets of Protein-Metabolite Interactions

ChemBioChem, 2009, 10(5), 799 - 801, doi:10.1002/cbic.200900010 published on 02.03.2009
ChemBioChem, online article
Saghatelian and colleagues recently introduced a global metabolite-profiling approach that allows protein-metabolite interactions (PMI) to be identified. This approach represents an excellent strategy and valuable tool for unraveling the many secrets of the metabolome. The key features of the methodology will be summarized here.

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Imaging of integrin alpha-v-beta-3 expression in patients with malignant glioma by [18F] Galacto-RGD positron emission tomography

Neuro-Oncology, 2009, 11(6), 861-870, doi:10.1215/15228517-2009-024 published on 01.03.2009
Neuro-Oncology, online article
Inhibitors targeting the integrin {alpha}vβ3 are promising new agents currently tested in clinical trials for supplemental therapy of glioblastoma multiforme (GBM). The aim of our study was to evaluate 18F-labeled glycosylated Arg-Gly-Asp peptide ([18F]Galacto-RGD) PET for noninvasive imaging of {alpha}vβ3 expression in patients with GBM, suggesting eligibility for this kind of additional treatment. Patients with suspected or recurrent GBM were examined with [18F]Galacto-RGD PET. Standardized uptake values (SUVs) of tumor hotspots, galea, and blood pool were derived by region-of-interest analysis. [18F]Galacto-RGD PET images were fused with cranial MR images for image-guided surgery. Tumor samples taken from areas with intense tracer accumulation in the [18F]Galacto-RGD PET images and were analyzed histologically and immunohistochemically for {alpha}vβ3 integrin expression. While normal brain tissue did not show significant tracer accumulation (mean SUV, 0.09 ± 0.04), GBMs demonstrated significant but heterogeneous tracer uptake, with a maximum in the highly proliferating and infiltrating areas of tumors (mean SUV, 1.6 ± 0.5). Immunohistochemical staining was prominent in tumor microvessels as well as glial tumor cells. In areas of highly proliferating glial tumor cells, tracer uptake (SUVs) in the [18F]Galacto-RGD PET images correlated with immunohistochemical {alpha}vβ3 integrin expression of corresponding tumor samples. These data suggest that [18F] Galacto-RGD PET successfully identifies {alpha}vβ3 expression in patients with GBM and might be a promising tool for planning and monitoring individualized cancer therapies targeting this integrin.

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Phosphorothioation of Oligonucleotides Strongly Influences the Inhibition of Bacterial (M.HhaI) and Human(Dnmt1) DNA Methyltransferases

ChemBioChem, 2009, 10, 728-734 published on 16.02.2009
ChemBioChem, online article
The cytidine analogue 5-fluoro-2-deoxycytidine (dCF) is a mechanism-based inhibitor of DNA methyltransferases. We report the synthesis of short 18-mer dsDNA oligomers containing a triple-hemimethylated CpG motive as a recognition sequence for the human methyltransferase Dnmt1. The DNA strands carry within these CpG islands dCF building blocks that function as mechanism-based inhibitors of the analyzed methyltransferases. In addition, we replaced the phosphodiester backbones at defined positions by phosphorothioates. These hypermodified DNA strands were investigated as inhibitors of the DNA methyltransferases M.HhaI and Dnmt1 in vitro. We could show that both methylases behave substantially differently in respect to the amount of DNA backbone modification.

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Structurally Refined b-Lactones as Potent Inhibitors of Devastating Bacterial Virulence Factors

ChemBioChem, 2009, doi: 10.1002/cbic.200800743, published on 10.02.2009
ChemBioChem, online article
With decreasing efficiency of antibiotic therapies against hospital- and community-acquired bacterial pathogens, the treatment of infectious diseases again represents a tremendous challenge for medicinal research. This challenge seems to be particularly difficult if one considers the sophisticated resistance strategies, which are effective against almost all currently available antibiotics, developed by bacterial pathogens, such as Staphylococcus aureus.

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Crystal structure analysis of DNA lesion repair and tolerance mechanisms

Curr. Opin. Struct. Biol., 2009, 19, 87-95 published on 04.02.2009
Curr. Opin. Struct. Biol., online article
Selective base pairing of the four canonical nucleobases is fundamental for the integrity of the genetic system. Information loss associated with DNA damage is a constant challenge and in response, organisms have evolved specialized defence systems consisting of DNA repair and lesion tolerance. DNA repair requires the action of different lesion recognition proteins such as lesion-specific glycosylases and DNA endonucleases. Lesion tolerance is established by special translesion synthesis (TLS) polymerases, which are able to bypass lesions during replication. In the past decade a large number of structures of repair proteins and TLS polymerases in complex with DNA containing individual lesions provided detailed insight into the chemistry of DNA repair and TLS. This review summarizes recent structural results.

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A Pan-Specific Inhibitor of the Polo-Box Domains of Polo-like Kinases Arrests Cancer Cells in Mitosis

ChemBioChem, 2009, 10.1002/cbic.200900059, published on 04.02.2009
ChemBioChem, online article
Polo-like kinases (Plks) are a conserved family of serine/threonine kinases.[1, 2] The family member Plk1 is a key regulator of mitosis[1, 2] and has been identified as a negative prognostic marker for tumor patients.[3, 4] The widespread recognition of Plk1 as a therapeutic target for the treatment of human tumors has triggered numerous drug discovery programs.[5–16] The vast majority of Plk1 inhibitors are thought to act on the ATP-binding pocket of the enzyme.[6–16] Due to the conserved nature of the ATP-binding pocket in the family of protein kinases and other ATP-binding enzymes, the development of monospecific, ATP-competitive inhibitors is an enormous challenge.

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Importin 8 Is a Gene Silencing Factor that Targets Argonaute Proteins to Distinct mRNAs

Cell, 2009, doi:10.1016/j.cell.2008.12.023, published on 22.01.2009
Cell, online article
Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading tomRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

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Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals

EMBO J., 2009, 28, 383-393 published on 15.01.2009
EMBO J., online article
DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error-free, and the third slow and error-prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase (pol), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two-polymerase combinations with pol dictate error-prone or error-free TLS across the same lesion. These results highlight the central role of pol in both error-prone and error-free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two-polymerase combinations.

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Formation of Organozincate Anions in LiCl-Mediated Zinc Insertion Reactions

Organometallics, 2009, 28, 3, 771-9 published on 06.01.2009
Organometallics, online article
Tetrahydrofuran solutions of the products formed in LiCl-mediated zinc insertion reactions into various organic halides RHal were analyzed by anion-mode electrospray ionization (ESI) mass spectrometry. In all cases, organozincate anions were observed. The reactions with RHal, Hal ) Br and I, yielded predominantly mononuclear complexes, such as ZnRHal2 - and ZnRHalCl-, whereas for the reaction with benzylchloride abundant polynuclear organozincates, such as Zn2Bn2Cl3 - and Zn3Bn3Cl4 -, were detected. The equilibria governing the stoichiometry and aggregation state of these complexes appear to be mainly controlled by the nature of the halide ions present in solution. It seems likely that the formation of organozincate complexes also changes the reactivity of the organozinc species, thus offering a rationale for the recently found pronounced effect of LiCl in organozinc chemistry. Additional preliminary studies suggest that organozincate anions as well as organozinc cations may moreover form in the absence of LiCl.

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Controlled Nucleation of DNA Metallization

Angew. Chem. Int. Ed., 2009, 48, 219-223, DOI:10.1002/anie.200803123 published on 28.11.2008
Angew. Chem. Int. Ed., online article
Silver-plated DNA: The deposit of a thin metal layer on biomolecules, such as DNA, requires the formation of small, magic-sized metal nuclei. Through the careful design of a reducing chemical functionality in the form of a dialdehyde, the nucleation process and thus the metallization step can be controlled.

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TU München
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