2010

Tissue Distribution of 5-Hydroxymethylcytosine and Search for Active Demethylation Intermediates

PLoS ONE, 2010, doi:10.1371/journal.pone.0015367, published on 23.12.2010
PLoS ONE, online article
5–Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5–formylcytosine (fC), 5-carboxylcytosine (caC), and 5–hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.

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Elucidation of the a-Keto-Aldehyde Binding Mechanism: A Lead Structure Motif for Proteasome Inhibition

Angewandte Chemie, 2010, DOI: 10.1002/anie.201005488, published on 19.12.2010

Angewandte Chemie, online article

The proteasome's participation in essential biological processes such as stress response, cell proliferation, apoptosis, and antigen presentation has been well established.[1] It is, therefore, not surprising that academia and the pharmaceutical industry have made efforts to develop a range of small synthetic inhibitors against this proteolytic molecular machine (see Scheme SS1 in the Supporting Information for examples).[2] An overall structural comparison of some wellcharacterized inhibitors[3] implies that most of these compounds form a covalent bond with the N-terminal nucleophilic threonine (Thr1)[4] located at the active sites in the two inner heptameric b rings of the 20S proteasome, termed b1, b2, and b5 according to the subunit of their origin.[5]

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Biosynthesis of Isoprenoids: Crystal Structure of the [4Fe–4S] Cluster Protein IspG

Journal of Molecular Biology,, 2010, doi:10.1016/j.jmb.2010.09.050, Volume 404, Issue 4, Pages 600-610 published on 10.12.2010
Journal of Molecular Biology, online article
IspG protein serves as the penultimate enzyme of the recently discovered non-mevalonate pathway for the biosynthesis of the universal isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The enzyme catalyzes the reductive ring opening of 2C-methyl-d-erythritol 2,4-cyclodiphosphate, which affords 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. The protein was crystallized under anaerobic conditions, and its three-dimensional structure was determined to a resolution of 2.7 Å. Each subunit of the c2 symmetric homodimer folds into two domains connected by a short linker sequence. The N-terminal domain (N domain) is an eight-stranded β barrel that belongs to the large TIM-barrel superfamily. The C-terminal domain (C domain) consists of a β sheet that is flanked on both sides by helices. One glutamate and three cysteine residues of the C domain coordinate a [4Fe–4S] cluster. Homodimer formation involves an extended contact area (about 1100 Å2) between helices 8 and 9 of each respective β barrel. Moreover, each C domain contacts the N domain of the partner subunit, but the interface regions are small (about 430 Å2). We propose that the enzyme substrate binds to the positively charged surface area at the C-terminal pole of the β barrel. The C domain carrying the iron–sulfur cluster could then move over to form a closed conformation where the substrate is sandwiched between the N domain and the C domain. This article completes the set of three dimensional structures of the non-mevalonate pathway enzymes, which are of specific interest as potential targets for tuberculostatic and antimalarial drugs.

 

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Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method

Analytical Biochemistry, 2010, doi:10.1016/j.ab.2010.12.004, published on 03.12.2010
Analytical Biochemistry, online article
The filter-aided sample preparation (FASP) method allows gel-free processing of biological samples sol- ubilized with detergents for proteomic analysis by mass spectrometry. In FASP detergents are removed by ultrafiltration, and after protein digestion peptides are separated from undigested material. Here we compare the effectiveness of different filtration devices for analysis of proteomes and glycoproteo- mes. We show that Microcon and Vivacon filtration units with nominal molecular weight cutoffs of 30,000 and 50,000 (30 and 50 k, respectively) are equally suitable for FASP, whereas Microcon 30 k units are most appropriate for mapping of N-glycosylation sites. The use of filters with these relatively large cutoffs facilitates depletion of detergents.

 

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Toward the Total Synthesis of Maoecrystal V: Establishment of Contiguous Quaternary Stereocenters

Org. Letters, 2010, DOI: 10.1021/ol102446u, 12 (24), pp 5656–5659 published on 18.11.2010
Org. Letters, online article
A synthetic strategy toward maoecrystal V has been identified. It has been shaped by the necessity to maneuver in sterically hindered molecular environments.

 

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Mechanism of replication blocking and bypass of Y-family polymerase Eta by bulky acetylaminofluorene DNA adducts

PNAS, 2010, vol. 107 no. 48 20720-20725, doi: 10.1073/pnas.1008894107 published on 12.11.2010

PNAS, online article

Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA and RNA synthesis. These lesions are consequently strong replication blocks. In addition bulky adducts give rise to point and frameshift mutations. The translesion synthesis (TLS) DNA polymerase Eta is able to bypass slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene- dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol Eta depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.

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Ferredoxin:NADPH oxidoreductase is recruited to thylakoids by binding to a polyproline type II helix in a pH-dependent manner

PNAS, 2010, doi: 10.1073/pnas.1009124107, vol. 107 no. 45 19260-19265 published on 09.11.2010
PNAS, online article
Ferredoxin:NADPH oxidoreductase (FNR) is a key enzyme of photosynthetic electron transport required for generation of reduction equivalents. Recently, two proteins were found to be involved in membrane-anchoring of FNR by specific interaction via a conserved Ser/Pro-rich motif: Tic62 and Trol. Our crystallographic study reveals that the FNR-binding motif, which forms a polyproline type II helix, induces self-assembly of two FNR monomers into a back-to-back dimer. Because binding occurs opposite to the FNR active sites, its activity is not affected by the interaction. Surface plasmon resonance analyses disclose a high affinity of FNR to the binding motif, which is strongly increased under acidic conditions. The pH of the chloroplast stroma changes dependent on the light conditions from neutral to slightly acidic in complete darkness or to alkaline at saturating light conditions. Recruiting of FNR to the thylakoids could therefore represent a regulatory mechanism to adapt FNR availability/activity to photosynthetic electron flow.

 

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Confident phosphorylation site localization using the Mascot Delta Score

Molecular & Cellular Proteomics, 2010, doi: 10.1074/mcp.M110.003830, published on 06.11.2010

Molecular & Cellular Proteomics, online article

Large scale phosphorylation analysis is more and more getting into focus of proteomic research. While it is now possible to identify thousands of phosphorylated peptides in a biological system, confident site localization remains challenging. Here we validate the Mascot Delta Score (MD score) as a simple method that achieves similar sensitivity and specificity for phosphosite localization as the published Ascore which is mainly used in conjunction with Sequest. The MD-score was evaluated using LC-MS/MS data of 180 individually synthesised phosphopeptides with precisely known phosphorylation sites. We tested the MD-score for a wide range of commonly available fragmentation methods and found it to be applicable throughout with high statistical significance. However, the different fragmentation techniques differ strongly in their ability to localise phosphorylation sites. At 1% FLR, the highest number of correctly assigned phosphopeptides was achieved by higher energy collision induced dissociation (HCD) in combination with an Orbitrap mass analyzer followed very closely by low resolution ion trap spectra obtained after electron transfer dissociation (ETD). Both these methods are significantly better than low resolution spectra acquired after collision induced dissociation (CID) and multi stage activation (MSA). Score thresholds determined from simple calibration functions for each fragmentation method were stable over replicate analyses of the phosphopeptide set. The MD-score outperforms the Ascore for tyrosine phosphorylated peptides and we further show that the ability to call sites correctly increases with increasing distance of two candidate sites within a peptide sequence. The MD-score does not require complex computational steps which makes it attractive in terms of practical utility. We provide all mass spectra and the synthetic peptides to the community so that the development of present and future localization software can be benchmarked and any laboratory can determine MD-scores and localization probabilities for their individual analytical set up.

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Residue-Specific Side-Chain Packing Determines the Backbone Dynamics of Transmembrane Model Helices

Biophysical Journal, 2010, doi:10.1016/j.bpj.2010.08.031, Volume 99, Issue 8, 2541-2549 published on 20.10.2010
Biophysical Journal, online article
The transmembrane domains (TMDs) of membrane-fusogenic proteins contain an overabundance of b-branched residues. In a previous effort to systematically study the relation among valine content, fusogenicity, and helix dynamics, we developed model TMDs that we termed LV-peptides. The content and position of valine in LV-peptides determine their fusogenicity and backbone dynamics, as shown experimentally. Here, we analyze their conformational dynamics and the underlying molecular forces using molecular-dynamics simulations. Our study reveals that backbone dynamics is correlated with the efficiency of side-chain to side-chain van der Waals packing between consecutive turns of the helix. Leu side chains rapidly interconvert between two rotameric states, thus favoring contacts to its i plusminus 3 and i plusminus 4 neighbors. Stereochemical restraints acting on valine side chains in the alpha-helix force both b-substituents into an orientation where i,i plusminus 3 interactions are less favorable than i, plusminus 4 interactions, thus inducing a local packing deficiency at VV3 motifs. We provide a quantitative molecular model to explain the relationship among chain connectivity, side-chain mobility, and backbone flexibility. We expect that this mechanism also defines the backbone flexibility of natural TMDs.

 

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Reductive Dehydroxylation of Allyl Alcohols by IspH Protein

Angewandte Chemie, 2010, DOI: 10.1002/anie.201000833, Volume 49, Issue 47, pages 8802–8809 published on 04.10.2010
Angewandte Chemie, online article
The biosynthesis of natural products is a treasure trove of unusual reaction mechanisms. This Minireview summarizes recent work on the structure and mechanism of IspH protein, which catalyzes the reductive dehydroxylation of an allyl alcohol in a biosynthetic pathway leading to isoprenoid precursors.

 

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Pharmacology of ionotropic glutamate receptors: A structural perspective

Bioorganic & Medicinal Chemistry, 2010, doi:10.1016/j.bmc.2010.09.012, 18 (2010) 7759–7772 published on 19.09.2010
Bioorganic & Medicinal Chemistry, online article
The impact of structural biology on the design of ligands (agonists, antagonists and modulators) for ionotropic glutamate receptors is reviewed.

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CIPSM spin-off AVIRU gets funding from the German Parliament

2010, published on 08.09.2010

LMU press release, www.aviru.de

CIPSM spin-off AVIRU stands for anti virulence and is a biotechnology start-up that is currently funded as an EXIST transfer of technology project by the German Federal Ministry of Economics and Technology in compliance with a resolution of the German Parliament. AVIRU aims to develop the proof-of-concept for an innovative anti-infective drug for the treatment of infections by multi-drug resistant bacterial pathogens.

www.aviru.de

LMU press release

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Synthesis and Antiplasmodial Activity of Highly Active Reverse Analogues of the Antimalarial Drug Candidate Fosmidomycin

ChemMedChem, 2010, DOI: 10.1002/cmdc.201000276, Volume 5, Issue 10, pages 1673–1676, published on 18.08.2010
ChemMedChem, online article
Although there are potent antimalarial drugs available, the spread of drug resistance has led to an increase in morbidity and mortality rates in malaria-endemic regions. To overcome these problems, new antimalarial drugs with novel modes of action have to be developed. The recently discovered nonmevalonate pathway of isoprenoid biosynthesis (MEP pathway) is an important metabolic pathway for the design of novel antimalarial drugs. Various pathogenic bacteria (e.g., Mycobacterium tuberculosis) and apicomplexan protozoa (e.g., Plasmodium falciparum) use the MEP pathway for the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are important precursors for the biosynthesis of various essential isoprenoids. In contrast to the latter microorganisms, humans produce IPP and DMAPP exclusively via the mevalonate–acetate pathway. The second enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) represents a validated target enzyme for the development of new antimalarial drugs. In 1998, the natural product fosmidomycin was identified as a potent inhibitor of P. falciparum DXR (PfDXR). The DXR catalyzes the rearrangement and reduction of 1-deoxy-d-xylulose 5-phosphate (DOXP) into 2-C-methyl-d-erythritol 4-phosphate (MEP). While the first step of the reaction requires a divalent cation (e.g., Mn2+ or Mg2+), the reduction step is NADPH-dependent. Clinical trials performed with fosmidomycin have already shown high efficiency in the treatment of acute, uncomplicated malaria tropica. However, the oral bioavailability of fosmidomycin is only moderate, with a resorption rate of about 10–30 %. Various research groups are involved in the design of novel DXR inhibitors. Molecular field analyses, as well as crystallographic studies, could already contribute in defining structural requirements for the development of potent DXR inhibitors. Consequently, different types of DXR inhibitors have been synthesized and biologically evaluated. Important structural elements for potent antimalarial activity are the hydroxamic acid functionality and the phosphonic acid group. The DXR–fosmidomycin complex crystal structure clearly revealed that the hydroxamic acid functionality chelates the divalent cation, while the phosphonic acid group occupies the phosphate binding site. Moreover, a defined distance between both functional groups is essential for potent antimalarial activity. Schlitzer and Klebe have shown that the two negative charges of the phosphonic acid group are necessary for high inhibitory activity. Furthermore, phosphonate prodrugs have been designed to improve the bioavailability of fosmidomycin (1) and FR900098 (2). A combinatorial approach for the synthesis of nonhydrolyzable phosphate mimics was reported by Link and co-workers, while Song recently described a coordination chemistry-based approach for the development of lipophilic DXR inhibitors that exhibit good to moderate activity against various pathogenic bacteria. To the best of our knowledge, a-aryl-substituted derivatives (4) of fosmidomycin are among the most active analogues known so far. Recently, Van Calenbergh reported that b-oxaisosters (3a,b) of fosmidomycin exhibit strong in vitro antimalarial activity. Interestingly, some of the new compounds contain an N-methyl-substituted hydroxamic acid group with a reverse orientation of the hydroxamate group (3b) compared to the lead compounds (1 and 2).

 

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20S Proteasome Inhibition: Designing Noncovalent Linear Peptide Mimics of the Natural Product TMC-95A

ChemMedChem,, 2010, DOI: 10.1002/cmdc.201000293, Volume 5, Issue 10, pages 1701–1705 published on 16.08.2010
ChemMedChem, online article
The ubiquitin-proteasome system (UPS) is responsible for the controlled degradation of proteins in eukaryotic cells through conjugation of ubiquitin molecules and subsequent cleavage by a multimeric protein complex known as the 26S proteasome. Proteolysis occurs within the 20S proteasome core particle (CP), a large barrel-shaped protein complex composed of four stacked rings made up of a and b subunits and which is found throughout all the three kingdoms of life. The a subunits constitute the top and bottom of the four rings and have substrate gating functions, whereas the b subunits, found within the two central rings of the complex, supply the catalytic machinery. In eukaryotes, this CP is composed of seven unique a subunits and seven unique b subunits, of which only three—b1, b2, and b5—possess a catalytic N-terminal threonine residue required for substrate hydrolysis (with chymotrypsin- like (CL), trypsin-like (TL), and peptidyl-glutamyl-peptide-hydrolyzing (PGPH) activities, respectively; see Supporting Information comment SC1).

 

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Total synthesis of newbouldine via reductive N - N bond formation

Tetrahedron, 2010, 66, 33, 6626 - 31 published on 14.08.2010
Tetrahedron, online article
The first total synthesis of newbouldine has been achieved employing a new, reductive N - N bond forming reaction. The asymmetric synthesis confirms that the natural product is a racemate.

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Biomimetic Synthesis of (−)-Pycnanthuquinone C through the Diels–Alder Reaction of a Vinyl Quinone

Angewandte Chemie, 2010, 49, 35, 6199 - 6202 published on 19.07.2010
Angewandte Chemie, online article
Diels–Alder reactions of vinyl quinones may provide a rapid entry to highly functionalized bi- and polycyclic ring systems. They involve the inverse-electron-demand cycloaddition of a suitable dienophile to a vinyl quinone, which presumably generates an “isoquinone methide” (Scheme 1). This reactive intermediate could then tautomerize in several ways to yield quinone methides, bicyclic quinones, or hydroquinones. If the isoquinone methide or quinone methide is intercepted by a nucleophile, such as water, a functionalized tetraline hydroquinone may result. This may be oxidized readily to the functionalized tetraline quinone.

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Quantification of the Sixth DNA Base Hydroxymethylcytosine in the Brain

Angewandte Chemie, 2010, 49, 31, 5375 - 77 published on 19.07.2010

Angewandte Chemie, online article

LC-MS has allowed the amount of the post-replicatively formed DNA base 5-hydroxymethylcytosine (see picture; left) to be quantified in brain tissue. The nucleoside is most abundant in areas that are associated with higher cognitive functions, and its content in mouse hippocampi seems to increase with age. The new method enables hydroxymethylcytosine to be quantified with unprecedented accuracy.

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Convergent Synthesis and Biological Evaluation of Syringolin A and Derivatives as Eukaryotic 20S Proteasome Inhibitors

European Journal of Organic Chemistry, 2010, DOI: 10.1002/ejoc.201090055, Volume 2010, Issue 21, page n/a published on 12.07.2010
European Journal of Organic Chemistry, online article
A convergent synthesis of SylA was developed and consists of the synthesis of a fully functionalized macrocycle, which is subsequently coupled with a urea moiety. For cyclization, ring-closing metathesis of a conformationally preorganized precursor was employed. The established synthetic route was then applied to the synthesis of SylA derivatives by using various peptidic side chains for decoration of the SylA macrocycle. The resulting collection of SylA analogues was tested for proteasome inhibition, revealing PEGylated SylA derivatives as the most potent proteasome inhibitors.

 

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Proteomics: a pragmatic perspective

Nature Biotechnology, 2010, doi:10.1038/nbt.1658, Pages: 695–709 published on 09.07.2010
Nature Biotechnology, online article
The evolution of mass spectrometry–based proteomic technologies has advanced our understanding of the complex and dynamic nature of proteomes while concurrently revealing that no ‘one-size-fits-all’ proteomic strategy can be used to address all biological questions. Whereas some techniques, such as those for analyzing protein complexes, have matured and are broadly applied with great success, others, such as global quantitative protein expression profiling for biomarker discovery, are still confined to a few expert laboratories. In this Perspective, we attempt to distill the wide array of conceivable proteomic approaches into a compact canon of techniques suited to asking and answering specific types of biological questions. By discussing the relationship between the complexity of a biological sample and the difficulty of implementing the appropriate analysis approach, we contrast areas of proteomics broadly usable today with those that require significant technical and conceptual development. We hope to provide nonexperts with a guide for calibrating expectations of what can realistically be learned from a proteomics experiment and for gauging the planning and execution effort. We further provide a detailed supplement explaining the most common techniques in proteomics.

 

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CIPSM spin-off XL-protein GmbH awarded with the 2010 Science4Life Venture Cup

2010, published on 07.07.2010
Science4Life press release

XL-protein

CIPSM spin-off XL-protein with its revolutionary PASylation technology has been awarded the Science4Life Venture Cup 2010 for its excellent business concept for the development of novel biologics with prolonged action. XL-biologics, a spin-off of XL-protein, will focus on the development of superior biopharmaceuticals with extended plasma half-life utilising the PASylation technology. Uli Binder, CTO of XL-protein, states: “We are very happy that our concept was so highly valued in this well reputed competition. This reassures our efforts to apply the ‘PASylation’ technology in order to help patients to improve their quality of life.” At the final stage of the contest XL-biologics successfully competed with business concepts by 54 teams mainly from the areas of biotechnology, pharmaceuticals and medical engineering. The Science4Life Venture Cup is the key business plan contest in Germany for life sciences and chemistry, co-sponsored by the states government of Hesse and Sanofi-Aventis.

 

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The 26S proteasome: assembly and function of a destructive machine

Trends in Biochemical Science, 2010, doi:10.1016/j.tibs.2010.05.005, Volume 35, Issue 11, Pages 634-642 published on 10.06.2010
Trends in Biochemical Science, online article
The heart of the ubiquitin-mediated degradation pathway, the 26S proteasome, endoproteolytically cleaves most intracellular proteins, thereby maintaining biological homeostasis and regulating many crucial processes in the cell. This hydrolyzing machine comprises more than 30 different subunits, which perform different functions including the recognition, unfolding, translocating and cleavage of protein substrates. Thus, careful assemblage and regulation of the 26S proteasome is essential to ensure correct positioning and function of each subunit, thereby preserving the delicate cellular balance between protein synthesis and degradation. Here, we review the most current research on the 26S proteasome assembly pathway, and describe the mechanism used by the cell to manage the complex structure and functions of the proteasome.

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CIPSM at the elementary school

In order to amaze our next generations for science and chemistry in particular, CIPSM hosted a two day event at an elementary school with lots of do-it-yourself experiments for the school kids. On the first day (11th of May) Oliver Baron gave an experimentary lecture with lots of explosions and "magical" reactions at the Department of Chemistry of the LMU for around 300 kids focusing on water and matter in general. The following day the kids experimented themselves and got a first glimpse into science. We at CIPSM got a very good feedback from the school kids which already franticly started buying chemistry kits.-)

 

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Proteome, Phosphoproteome, and N-Glycoproteome Are Quantitatively Preserved in Formalin-Fixed Paraffin-Embedded Tissue and Analyzable by High-Resolution Mass Spectrometry

J. Proteome Research, 2010, DOI: 10.1021/pr100234w, 9 (7), pp 3688–3700 published on 16.05.2010
J. Proteome Research, online article
Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement timesthe same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or the peptide level. Application of our FFPE-FASP protocol to phosphorylation and N-glycosylation pinpointed nearly 5000 phosphosites and 1500 N-glycosylation sites. Analysis of FFPE tissue of the SILAC mouse revealed that these post-translational modifications were quantitatively preserved. Thus, FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications.

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Exzellenzinitiative für die Grundschule - CIPSM unterrichtet Grundschüler

2010, published on 12.05.2010
Grundschule an der Würm Stockdorf
CIPSM hosts a two day event at an elementary school with lots of do-it-yourself experiments for the school kids. On the first day (11th of May) Oliver Baron gives a experimentary lecture at the Department of Chemistry of the LMU for around 300 kids focusing on water and matter in general. The following day the kids can experiment themselves and get a first glimpse into science.

 

Exzellenzinitiative für die Grundschule

 

München, 11.–12. Mai 2010 – Münchner Exzellenzcluster startet Projekt mit Grundschülern: Ziel dieses gemeinsamen Projektes zwischen dem auf dem Gebiet der Proteinwissenschaften forschenden Münchner Exzellenzclusters CIPSM und der Grundschule an der Würm Stockdorf ist, Grundschulkinder wissenschaftliche Zusammenhänge anschaulich und spannend zu vermitteln um sie frühzeitig für die Natur bzw. deren Wissenschaften zu sensibilisieren. Denn: Es kann nicht schaden Kinder auf wichtige Ereignisse und Phänomene in unserer Welt Neugierig zu machen und ihr Interesse für die Vielfalt naturwissenschaftlicher Themen zu wecken.

Im Rahmen dieses gemeinsamen Projektes rund um das Thema „Wasser“ als Lebenselixier für alle Lebewesen sollen einige Fragen beantwortet werden: Aus was besteht Wasser, was sind seine Eigenschaften, wie wird Trinkwasser gewonnen, wodurch wird es verschmutzt und was passiert mit dem Abwasser? Wer oder was reinigt das Abwasser? Gibt es im Abwasser Lebewesen, kann man das Wasser der Würm trinken? ….und vieles mehr. Solche Kinderfragen wirken auf den ersten Blick trivial. Tatsächlich sind sie jedoch nicht für jeden leicht zu beantworten, denn dahinter steckt reichliches naturwissenschaftliches Wissen.

 

Als Vorbereitung zum Projekttag am 12. Mai 2010 an der „Grundschule an der Würm“ in Stockdorf, Landkreis Starnberg, wird eine kindgerechte und anschauliche chemische Experimentalvorlesung am 11. Mai 2010 für alle Schüler der Jahrgangsstufen 1 – 4 am HighTechCampus Großhadern des Departments Chemie der Ludwig-Maximilians-Universität München veranstaltet.

 

Hierbei wird auf wissenschaftliches Hinterfragen aufmerksam gemacht: Was ist Materie? Wie ist sie aufgebaut? Was sind Moleküle? Wie können sie reagieren? In dieser Vorlesung werden die Kinder über Experimente an einfache Erklärungen zum Aufbau der Materie herangeführt.

 

Der 12. Mai 2010 steht ganz unter dem Motto „selber ausprobieren“. Es stehen insgesamt 13 Versuchsanordnungen in der Grundschule an der Würm Stockdorf den Schülern zum eigenen Experimentieren zur Auswahl. Die einzelnen Klassen werden in kleinen Gruppen verschiedenste Versuche unter Anleitung der Wissenschaftler des Exzellenzclusters und der Lehrkräfte der Schule durchführen, wobei der Schwierigkeitsgrad den Jahrgangsstufen entsprechend angepasst wird.

 

 

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Thomas Carell was honored with the Cross of Merit on ribbon of the Federal Republic of Germany

2010, published on 04.05.2010
Bayerische Staatsregierung
CIPSM speaker Thomas Carell was honored today by the Bavarian Prime Minister Horst Seehofer with the Cross of Merit on ribbon of the Federal Republic of Germany.

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Showdomycin as a Versatile Chemical Tool for the Detection of Pathogenesis-Associated Enzymes in Bacteria

JACS, 2010, DOI: 10.1021/ja909150y, pp 6964–6972 published on 30.04.2010
JACSonline article
Showdomycin is a potent nucleoside antibiotic that displays a high structural similarity to uridine and pseudouridine. No detailed target analysis of this very unusual electrophilic natural product has been carried out so far. To unravel its biological function, we synthesized a showdomycin probe that can be appended with a fluorophor or a biotin marker via click chemistry and identified diverse enzymes which were important for either the viability or virulence of pathogenic bacteria. Our results indicate that the antibiotic effect of showdomycin against Staphylococcus aureus may be due to the inhibition of various essential enzymes, especially MurA1 and MurA2, which are required for cell wall biosynthesis. Although real-time polymerase chain reaction revealed that the MurA2 gene was expressed equally in four S. aureus strains, our probe studies showed that MurA2 was activated in only one multiresistant S. aureus strain, and only this strain was resistant to elevated concentrations of the MurA inhibitor fosfomycin, suggesting its potential role as an antibiotic bypass mechanism in the case of MurA1 inhibition. Moreover, we utilized this tool to compare enzyme profiles of different pathogenic strains, which provided unique insights in regulatory differences as well as strain-specific signatures.

 

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Brain Phosphoproteome Obtained by a FASP-Based Method Reveals Plasma Membrane Protein Topology

J. Proteome Research, 2010, DOI: 10.1021/pr1002214, 9 (6), pp 3280–3289 published on 26.04.2010
J. Proteome Research, online article
Taking advantage of the recently developed Filter Assisted Sample Preparation (FASP) method for sample preparation, we performed an in-depth analysis of phosphorylation sites in mouse brain. To maximize the number of detected phosphorylation sites, we fractionated proteins by size exclusion chromatography (SEC) or separated tryptic peptides on an anion exchanger (SAX) prior or after the TiO2-based phosphopeptide enrichment, respectively. SEC allowed analysis of minute tissue samples (1 mg total protein), and resulted in identification of more than 4000 sites in a single experiment, comprising eight fractions. SAX in a pipet tip format offered a convenient and rapid way to fractionate phosphopeptides and mapped more than 5000 sites in a single six fraction experiment. To enrich peptides containing phosphotyrosine residues, we describe a filter aided antibody capturing and elution (FACE) method that requires only the uncoupled instead of resin-immobilized capture reagent. In total, we identified 12 035 phosphorylation sites on 4579 brain proteins of which 8446 are novel. Gene Ontology annotation reveals that 23% of indentified sites are located on plasma membrane proteins, including a large number of ion channels and transporters. Together with the glycosylation sites from a recent large-scale study, they can confirm or correct predicted membrane topologies of these proteins, as we show for the examples calcium channels and glutamate receptors.

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Oxidation State, Aggregation, and Heterolytic Dissociation of Allyl Indium Reagents

JACS, 2010, 132 (17), 6032–6040 published on 08.04.2010
JACS, online article
Solutions of allyl indium reagents formed in the reactions of indium with allyl bromide and allyl iodide, respectively, in N,N-dimethylformamide, tetrahydrofuran, and water were analyzed by a combination of electrospray-ionization mass spectrometry, temperature-dependent 1H NMR spectroscopy, and electrical conductivity measurements. Additional mass spectrometric experiments probed charge-tagged derivatives of the allyl indium reagents. The results obtained indicate the presence of allyl indium(+3) species, which undergo heterolytic dissociation to yield ions such as InR2(solv)+ and InRX3 - with R ) allyl and X ) Brand I. The extent of dissociation is greatest for N,N-dimethylformamide, whereas aggregation effects are more pronounced for the less polar tetrahydrofuran. The heterolytic dissociation of the allyl indium reagents supposedly enhances their reactivity by simultaneously providing highly Lewis acidic allyl indium cations and nucleophilic allyl indate anions.

koszinowski_jacs_2010_500



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Crystal Structure of a Cisplatin-(1,3-GTG) Cross-Link within DNA Polymerase eta

Angew. Chem. Intl. Ed., 2010, 49 (17), 3077-3080, doi: 10.1002/anie.201000414 published on 23.03.2010
Angewandte Chemie International Edition, online article
Flipped out: The cisplatin-(1,3-GTG) adduct, a strongly cytotoxic DNA lesion generated by the action of cisplatin therapeutics, is partially bypassed by Y-family DNA polymerase . Crystal structure data on this DNA-protein complex explain the partial-bypass process. The central dT unit of the lesion is turned out and blocks the movement of the enzyme along the DNA duplex.

Carell_Angewandte2010_500



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Natural Products and Their Biological Targets: Proteomic and Metabolomic Labeling Strategies

Angewandte Chemie, 2010, Volume 49, Issue 15, pages 2680–2698, DOI: 10.1002/anie.200905352; pages 2680–2698 published on 23.03.2010
Angewandte Chemie, online article
Activity-based protein profiling (ABPP) has matured into a standard method for the fast, sensitive, and selective identification of enzyme activity and inhibitors in proteomes. By using natural product based probes, the targets of many uncharacterized molecules can be easily identified in complex proteomes, and their exact function and mechanism of action understood. Natural products and their derivatives can also serve as pharmaceutical lead structures that impede essential components in the cell and their effects can be studied in biological assays. Since the complex regulatory processes in a cell go beyond mere transcription, translation, and activation, it is imperative to also identify the products of the active proteome—the metabolites and binding partners of individual enzymes and proteins. Therefore, methods by which the chemically complex metabolome can be characterized are necessary. A series of interesting approaches have become available in recent years that enable the global investigation of enzyme–metabolite pairs.

 

sieber_angew_2010_500



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DNA (6-4) Photolyases Reduce Dewar Isomers for Isomerization into (6-4) Lesions

J. Am. Chem. Soc., 2010, 132(10), 3254–3255, doi: 10.1021/ja910917f published on 18.02.2010
Journal of the American Chemical Society, online article
Repair of the Dewar valence isomers by (6-4) photolyases proceeds via an enzyme catalyzed ring-opening reaction of the Dewar lesion to the (6-4) photoproduct.

Carell_JACS2010_500



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Confident phosphorylation site localization using the Mascot Delta Score

Chemistry A European Joournal, 2010, 16, 11, 3285 - 90 published on 18.02.2010
Chemistry - A European Journal, online article
Small metal clusters fill the gap between the atomic scale and the metallic state with its distinctive bulk phenomena.[1] Besides being of high fundamental interest, this intermediate character of metal clusters also gives rise to unique and potentially useful electronic, magnetic, and optical properties.[ 2] To bring these properties to real-world applications, however, the clusters must be stabilized and prevented from spontaneous aggregation and other decomposition reactions. One successful strategy relies on the immobilization of metal clusters on surfaces.[3] Another approach stabilizes the clusters by the attachment of protective ligands.[4] Quite often the same ligands can also serve as templates for the formation of the metal clusters.[4] This favorable situation is realized for a manifold of silver nanoclusters, which are synthesized by the reduction of silver salts in the presence of templates, such as poly(amidoamine) dendrimers,[5] poly- (acrylic acid) derivatives,[6] poly(methacrylic acid),[7] peptides,[8] or DNA.[9] The thus formed nanoclusters contain only a few Ag atoms and hence exhibit molecule-like properties,[ 10] in contrast to the conventional larger nanoparticles, which more closely resemble the metallic state.[11] In particular, the strong fluorescence of silver nanoclusters has attracted significant attention because of its potential practical applications.[5–10] Among the various species investigated so far, silver nanoclusters encapsulated by the single-stranded oligonucleotide dC12 arguably constitute the most promising system thanks to their very high photoemission rates and their excellent photostability.[12]

koszinowski_chemeur_2010_500



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A Highly Charged Ag6 4+ Core in a DNA-Encapsulated Silver Nanocluster

Chemistry A European Joournal, 2010, 16, 11, 3285 - 90 published on 18.02.2010
Chemistry - A European Journal, online article
Small metal clusters fill the gap between the atomic scale and the metallic state with its distinctive bulk phenomena.[1] Besides being of high fundamental interest, this intermediate character of metal clusters also gives rise to unique and potentially useful electronic, magnetic, and optical properties.[ 2] To bring these properties to real-world applications, however, the clusters must be stabilized and prevented from spontaneous aggregation and other decomposition reactions. One successful strategy relies on the immobilization of metal clusters on surfaces.[3] Another approach stabilizes the clusters by the attachment of protective ligands.[4] Quite often the same ligands can also serve as templates for the formation of the metal clusters.[4] This favorable situation is realized for a manifold of silver nanoclusters, which are synthesized by the reduction of silver salts in the presence of templates, such as poly(amidoamine) dendrimers,[5] poly- (acrylic acid) derivatives,[6] poly(methacrylic acid),[7] peptides,[8] or DNA.[9] The thus formed nanoclusters contain only a few Ag atoms and hence exhibit molecule-like properties,[ 10] in contrast to the conventional larger nanoparticles, which more closely resemble the metallic state.[11] In particular, the strong fluorescence of silver nanoclusters has attracted significant attention because of its potential practical applications.[5–10] Among the various species investigated so far, silver nanoclusters encapsulated by the single-stranded oligonucleotide dC12 arguably constitute the most promising system thanks to their very high photoemission rates and their excellent photostability.[12]

 



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Structural features of fusogenic model transmembrane domains that differentially regulate inner and outer leaflet mixing in membrane fusion

Molecular Membrane Biology, 2010, DOI: 10.3109/09687680903362044, Volume 27, pp. 1-11(11) published on 01.01.2010
Molecular Membrane Biology, online article
The transmembrane domains of fusion proteins areknownto beimportant for their fusogenic activity. Inaneffort to systematically investigate the structure/function relationships of transmembrane domains we had previously designed LV-peptides that mimic natural fusion proteinTMDsin their ability to drive fusion after incorporation into liposomalmembranes. Here,weinvestigate the impact of different structural features of LV-peptide TMDs on inner and outer leaflet mixing. We find that fusion driven by the helical peptides involves a hemifusion intermediate as previously seen for natural fusion proteins. Helix backbone dynamics enhances fusion by selectively promoting outer leaflet mixing. Furthermore, the hydrophobic length of the peptides as well as covalent attachment of long acyl chains affects outer and inner leaflet mixing to different extents. Different structural features of transmembrane domains thus appear to differentially influence the rearrangements of lipids in fusion initiation and the hemifusion-to-fusion transition. The relevance of these findings in respect to the function of natural fusion proteins is discussed.

Mol_Mem_Biol_2010_500



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Probing the reaction mechanism of IspH protein by x-ray structure analysis

PNAS, 2010, 107, 3, 1077 - 81 published on 18.01.2008
PNAS, online article
Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovered deoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

groll_pnas_10_500



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LMUexcellent
TU München
MPG
Helmholz Muenchen
MPI of Neurobiology
MPI of Biochemistry