Content
2012
The radical SAM enzyme spore photoproduct lyase employs a tyrosyl radical for DNA repair
28.11.2012
Chemical Communications,
2012,
DOI: 10.1039/C2CC37735G,
49, 722-724
published on 28.11.2012
The spore photoproduct lyase is a radical SAM enzyme, which repairs 5-(α-thyminyl)-5,6-dihydrothymidine. Here we show that the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction.
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Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 30-splice site recognition
21.11.2012
Nucleic Acids Research,
2012,
doi:10.1093/nar/gks1097,
Vol. 41, No. 2 ,1343–1354
published on 21.11.2012
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Recognition of the 30-splice site is a key step in premRNA
splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65UHM) reveals that, in addition to the known U2AF65UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82- Pro83 motif rigidifies a long unstructured linker
in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 30-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA
complex.
Synthesis of ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine containing histone H3 using the pyrrolysine system
15.11.2012
Chem. Commun.,,
2012,
DOI: 10.1039/C2CC37836A,
2013,49, 379-381
published on 15.11.2012
Recently new lysine modifications were detected in histones and other proteins. Using the pyrrolysine amber suppression system we genetically inserted three of the new amino acids ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine site specifically into histone H3. The lysine at position 9 (H3 K9), which is known to be highly modified in chromatin, was replaced by these unnatural amino acids.
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Isotope-Based Analysis of Modified tRNA Nucleosides Correlates Modification Density with Translational Efficiency
04.10.2012
Angew. Chem.,
2012,
DOI: 10.1002/anie.201203769,
Volume 51, Issue 44, pages 11162–11165,
published on 04.10.2012
Transfer RNAs (tRNAs) are adapter molecules needed to translate genetic information into a peptide sequence. At the ribosome, the anticodon of each tRNA reads the corresponding codon of the messenger RNA. This anticodon– codon interaction allows the ribosome_s large subunit to catalyze amide-bond formation between the cognate amino acids present at the 3’ terminus of aminoacyl-tRNAs and the growing peptide chain. The tRNA adapters required for this process display a surprisingly large chemical diversity. Aside from the four canonical nucleosides A, C,G, and U, more than 100 modified nucleosides are key constituents. The most diverse and complex chemical structures are found in the anticodon stem-loop either in the anticodon at the wobble position or directly adjacent to the 3’ position of the anticodon, suggesting that here the chemical complexity is necessary to establish translational fidelity. The ribosome seems to need the modified anticodon region to better distinguish correctly base-paired tRNA from mispaired interactions in order to prohibit, for example, codon-slippage processes that would lead to frameshifts.
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A Simple and Effective Cleavable Linker for Chemical Proteomics Applications Yinliang Yang,
01.10.2012
MCP,
2012,
doi: 10.1074/mcp.M112.021014 mcp.M112.021014.,
published on 01.10.2012
ABPP, activity-based protein profiling; ABP, activity-based probe; Cat, Cathepsin; DIC, diisopropylcarbodiimide; DIEA, N,N-diisopropyl-N-ethylamine; DMEM, dulbecco's modified eagle medium; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, hydroxybenzotriazole; MHC, major histocompatibility complex; SB, sample buffer; SPPS, solid phase peptide synthesis; TBTA, tris- (benzyltriazolylmethyl)amine; TCEP, tris-(2-carboxyethyl)phosphine; TEV, tobacco etch virus.
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Deactivation of the E. coli pH Stress Sensor CadC by Cadaverine
17.09.2012
Journal of Molecular Biology,
2012,
http://dx.doi.org/10.1016/j.jmb.2012.08.023,,
Volume 424, Issues 1–2, Pages 15–27
published on 17.09.2012
Journal of Molecular Biology, online article
Journal of Molecular Biology, online article
At acidic pH and in the presence of lysine, the pH sensor CadC activates transcription of the cadBA operon encoding the lysine/cadaverine antiporter CadB and the lysine decarboxylase CadA. In effect, these proteins contribute to acid stress adaptation in Escherichia coli. cadBA expression is feedback inhibited by cadaverine, and a cadaverine binding site is predicted within the central cavity of the periplasmic domain of CadC on the basis of its crystallographic analysis. Our present study demonstrates that this site only partially accounts for the cadaverine response in vivo. Instead, evidence for a second, pivotal binding site was collected, which overlaps with the pH-responsive patch of amino acids located at the dimer interface of the periplasmic domain. The temporal response of the E. coli Cad module upon acid shock was measured and modeled for two CadC variants with mutated cadaverine binding sites. These studies supported a cascade-like binding and deactivation model for the CadC dimer: binding of cadaverine within the pair of central cavities triggers a conformational transition that exposes two further binding sites at the dimer interface, and the occupation of those stabilizes the inactive conformation. Altogether, these data represent a striking example for the deactivation of a pH sensor.
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Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells
11.09.2012
Molecular Systems Biology,
2012,
doi:10.1038/msb.2012.40,
published on 11.09.2012
Molecular Systems Biology, online article
Molecular Systems Biology, online article
Extra chromosome copies markedly alter the physiology of eukaryotic cells, but the underlying reasons are not well understood. We created human trisomic and tetrasomic cell lines and determined the quantitative changes in their transcriptome and proteome in comparison with their diploid counterparts. We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels. Furthermore, using the quantitative data we investigated the changes of cellular pathways in response to aneuploidy. This analysis revealed specific and uniform alterations in pathway regulation in cells with extra chromosomes. For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated. In particular, we found that the p62-dependent selective autophagy is activated in the human trisomic and tetrasomic cells. Our data present the first broad proteomic analysis of human cells with abnormal karyotypes and suggest a uniform cellular response to the presence of an extra chromosome.
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A Conformationally Frozen Peptoid Boosts CXCR4 Affinity and Anti-HIV Activity
06.08.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201202090,
Volume 51, Issue 32, pages 8110–8113
published on 06.08.2012
There can be only one: Using a peptoid motif obtained by shifting the arginine side chain of a pentapeptide previously developed by Fujii et al. to the neighboring nitrogen atom restricts the conformational freedom and yields a conformationally homogeneous peptide (see picture) with a 100-fold higher binding affinity to the chemokine receptor CXCR4 in the picomolar range. Its efficiency to inhibit HIV-1 infections is also demonstrated.
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Homotypic Interaction and Amino Acid Distribution of Unilaterally Conserved Transmembrane Helices
13.07.2012
Journal of Molecular Biology,
2012,
doi:10.1016/j.jmb.2012.04.008,
Pages 251–257
published on 13.07.2012
Formation of non-covalent functional complexes of integral membrane proteins is frequently supported by sequence-specific interaction of their transmembrane helices. Here, we aligned human single-span membrane proteins with orthologs from other eukaryotes. We find that almost half of the human single-span membrane proteins contain a transmembrane helix that exhibits significant non-random unilateral conservation. Furthermore, unilateral conservation of transmembrane domains (TMDs) correlates well with their ability to self-interact. Glycine, polar non-ionizable, and aromatic amino acids are overrepresented in conserved versus non-conserved helix faces. Hence, our genome-wide analysis indicates that these amino acid types generally support interaction of single-span membrane protein TMDs.
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Structural insights into recognition and repair of UV-DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme
02.07.2012
Nucleic Acids Research,
2012,
doi:10.1093/nar/gks603,
1–11
published on 02.07.2012
Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase) that repairs a specific UV-induced DNA lesion, the spore photoproduct (SP), through an unprecedented radical-based mechanism. Unlike DNA photolyases, SP lyase belongs to the emerging superfamily of radical S-adenosyl-l-methionine (SAM) enzymes and uses a [4Fe–4S]1+ cluster and SAM to initiate the repair reaction. We report here the first crystal structure of this enigmatic enzyme in complex with its [4Fe–4S] cluster and its SAM cofactor, in the absence and presence of a DNA lesion, the dinucleoside SP. The high resolution structures provide fundamental insights into the active site, the DNA lesion recognition and binding which involve a β-hairpin structure. We show that SAM and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the α-thyminyl radical moiety, respectively. Based on structural and biochemical characterizations of mutant proteins, we substantiate the role of this cysteine in the enzymatic mechanism. Our structure reveals how SP lyase combines specific features of radical SAM and DNA repair enzymes to enable a complex radical-based repair reaction to take place.
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Structure and Function of Noncanonical Nucleobases
28.06.2012
Angewandte Chemie,
2012,
DOI: 10.1002/ange.201201193,
Volume 51, Issue 29, pages 7110–7131,
published on 28.06.2012
DNA and RNA contain, next to the four canonical nucleobases,
a number of modified nucleosides that extend their chemical information content. RNA is particularly rich in modifications, which is obviously an adaptation to their highly complex and variable functions. In fact, the modified nucleosides and their chemical structures establish a second layer of information which is of central importance to the function of the RNA molecules. Also the chemical diversity of DNA is greater than originally thought. Next to the four canonical bases, the DNA of higher organisms contains a total of four epigenetic bases: m5dC, hm5dC, f5dC und ca5dC. While all cells of an organism contain the same genetic material, their vastly different function and properties inside complex higher organisms require the controlled silencing and activation of cell-type specific genes. The regulation of the underlying silencing and activation process requires an additional layer of epigenetic information, which is clearly linked to increased chemical diversity. This diversity is provided by the modified noncanonical nucleosides in both DNA and RNA.
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CIPSM selected as Cluster of Excellence once more... YEAH!
15.06.2012
published on 15.06.2012
Thank you! CIPSM has once more been selected as a Cluster of Excellence in the German Excellence Initiative.
In the next 5 years CIPSM will try to make sure, that it is here to stay. We thank all the people that helped to make that possible.
More information can be found here
Dynamics of ultraviolet-induced DNA lesions: Dewar formation guided by pre-tension induced by the backbone
12.06.2012
New Journal of Physics,
2012,
doi:10.1088/1367-2630/14/6/065006,
14
published on 12.06.2012
New Journal of Physics, online article
New Journal of Physics, online article
The photophysical and photochemical processes driving the formation of the ultraviolet (UV)-induced DNA Dewar lesion from the T(6-4)T dimer are investigated by time-resolved spectroscopy and quantum chemical modelling. Time-resolved absorption and emission spectroscopy in the UV revealed a biexponential decay of the electronically excited state (S1) with time constants in the 100 ps and 1 ns range. From the S1 state the system forms the Dewar lesion (proven by time-resolved infrared spectroscopy), the triplet state of the T(6-4)T dimer and the ground state of the original T(6-4)T dimer. The decay process from the excited singlet is activated and thus temperature dependent. Quantum chemical modelling is used to describe the reaction path via a minimum on the excited electronic potential energy surface in close proximity to a triplet state. The transition to the Dewar isomer competes with internal conversion and with triplet formation. Only if the backbone between the two thymines is closed, is the Dewar isomer formed with a significant yield. The simulations reveal that the tension built up by the backbone is required for guiding the reaction to the conical intersection leading to the Dewar isomer.
Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix
11.06.2012
Protein Science,
2012,
DOI: 10.1002/pro.2094,
Volume 21, Issue 7, pages 1097–1102
published on 11.06.2012
The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins.
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Discovery of the HSP90 regulated proteome by CIPSM scientists featured on the cover of Mol Cell Proteomics
01.06.2012
Molecular & Cellular Proteomics,
2012,
doi: 10.1074/mcp.M111.016675,
published on 01.06.2012
HSP90 is a central player in the folding and maturation of many proteins. More than two hundred HSP90 clients have been identified by classical biochemical techniques including important signaling proteins with high relevance to human cancer pathways. HSP90 inhibition has thus become an attractive therapeutic concept and multiple molecules are currently in clinical trials. It is therefore of fundamental biological and medical importance to identify, ideally, all HSP90 clients and HSP90 regulated proteins. To this end, we have taken a global and a chemical proteomic approach in geldanamycin treated cancer cell lines using stable isotope labeling with amino acids in cell culture and quantitative mass spectrometry. We identified >6200 proteins in four different human cell lines and ∼1600 proteins showed significant regulation upon drug treatment. Gene ontology and pathway/network analysis revealed common and cell-type specific regulatory effects with strong connections to unfolded protein binding and protein kinase activity. Of the 288 identified protein kinases, 98 were geldanamycin treatment including >50 kinases not formerly known to be regulated by HSP90. Protein turn-over measurements using pulsed stable isotope labeling with amino acids in cell culture showed that protein down-regulation by HSP90 inhibition correlates with protein half-life in many cases. Protein kinases show significantly shorter half lives than other proteins highlighting both challenges and opportunities for HSP90 inhibition in cancer therapy. The proteomic responses of the HSP90 drugs geldanamycin and PU-H71 were highly similar suggesting that both drugs work by similar molecular mechanisms. Using HSP90 immunoprecipitation, we validated several kinases (AXL, DDR1, TRIO) and other signaling proteins (BIRC6, ISG15, FLII), as novel clients of HSP90. Taken together, our study broadly defines the cellular proteome response to HSP90 inhibition and provides a rich resource for further investigation relevant for the treatment of cancer.
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Mechanism and Stem-Cell Activity of 5-Carboxycytosine Decarboxylation Determined by Isotope Tracing
29.05.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201202583,
Volume 51, Issue 26, pages 6516–6520,
published on 29.05.2012
5-Methylcytosine (mC) is an important, well-known nucleobase
modification that is involved in many biological processes,
including gene expression, genomic imprinting, Xchromosome
inactivation, and suppression of transposable elements.
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ONIOM approach for non-adiabatic on-the-fly molecular dynamics demonstrated for the backbone controlled Dewar valence isomerization
25.05.2012
Journal of Chemical Physics,
2012,
http://dx.doi.org/10.1063/1.4720090,
Volume 136; Issue 20; Pages 10
published on 25.05.2012
Journal of Chemical Physics, online article
Journal of Chemical Physics, online article
Non-adiabatic on-the-fly molecular dynamics (NA-O-MD) simulations require the electronic wavefunction, energy gradients, and derivative coupling vectors in every timestep. Thus, they are commonly restricted to the excited state dynamics of molecules with up to ≈20 atoms. We discuss an approximation that combines the ONIOM(QM:QM) method with NA-O-MD simulations to allow calculations for larger molecules. As a proof of principle we present the excited state dynamics of a (6-4)-lesion containing dinucleotide (63 atoms), and especially the importance to include the confinement effects of the DNA backbone. The method is able to include electron correlation on a high level of theory and offers an attractive alternative to QM:MM approaches for moderate sized systems with unknown force fields.
A Genetically Encoded Norbornene Amino Acid for the Mild and Selective Modification of Proteins in a Copper-Free Click Reaction
27.04.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201109252,
Volume 51, Issue 18, pages 4466–4469
published on 27.04.2012
Methods for the site-specific chemical modification of proteins
are currently of immense importance for the synthesis of
protein–hybrid compounds for pharmaceutical and diagnostic
purposes. Most of the methods rely on the reaction of free
protein thiols with maleimides or the reaction of lysine side
chains with activated esters. These methods provide only
limited specificity, which is prompting researchers to develop
alternative strategies that involve the incorporation of special
unnatural amino acid into proteins to enable site-specific
bioorthogonal functionalization. Among the developed
methods, the CuI-catalyzed reaction of a protein containing
an alkyne amino acid with azides stands out as the most
thoroughly investigated technology. However, the need for
CuI salts, which may harm the protein structure, limits the
technology. This fuels current interest to develop copperfree
coupling reactions that are compatible with fragile
protein structures. Here we show that these requirments
can be met with a specially encoded norbornene amino acid
which reacts selectively with nitrile imines.
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Mechanism of Translesion Transcription by RNA Polymerase II and Its Role in Cellular Resistance to DNA Damage
13.04.2012
Molecular Cell,
2012,
DOI 10.1016/j.molcel.2012.02.006,
46,18–29,
published on 13.04.2012
UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge
helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.
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H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization
29.03.2012
Nucleic Acids Research,
2012,
doi:10.1093/nar/gks267,
2012, 1–14
published on 29.03.2012
Nucleic Acids Research, online article
Nucleic Acids Research, online article
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.
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The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA
15.03.2012
Nucleic Acids Research,
2012,
doi: 10.1093/nar/gks240,
published on 15.03.2012
The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer’s disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i6A) into 2-methylthio-N6-isopentenyladenosine (ms2i6A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.
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The 19S Cap Puzzle: A New Jigsaw Piece
07.03.2012
Cell,
2012,
DOI 10.1016/j.str.2012.02.006,
Volume 20, Issue 3, 387-388
published on 07.03.2012
After elucidation of the atomic details of 20S proteasomes, current research focuses on the regulatory 19S particle. In this issue of Structure, He et al. present the crystal structure of Rpn2 and use electron microscopy to examine differences between Rpn2 and Rpn1.
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Radiolabelled RGD peptides for imaging and therapy
03.03.2012
Nuclear Medicine & Molecular Imaging,
2012,
DOI 10.1007/s00259-011-2028-1,
Volume 39, Issue 1 Supplement, pp 126-138
published on 03.03.2012
Imaging of angiogenesis has become increasingly important with the rising use of targeted antiangiogenic therapies like bevacizumab (Avastin). Non-invasive assessment of angiogenic activity is in this respect interesting, e.g. for response assessment of such targeted antiangiogenic therapies. One promising approach of angiogenesis imaging is imaging of specific molecular markers of the angiogenic cascade like the integrin αvβ3. For molecular imaging of integrin expression, the use of radiolabelled peptides is still the only approach that has been successfully translated into the clinic. In this review we will summarize the current data on imaging of αvβ3 expression using radiolabelled RGD peptides with a focus on tracers already in clinical use. A perspective will be presented on the future clinical use of radiolabelled RGD peptides including an outlook on potential applications for radionuclide therapy
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Rapid optical control of nociception with an ion-channel photoswitch
19.02.2012
Nature Methods,
2012,
doi:10.1038/nmeth.1897,
published on 19.02.2012
Local anesthetics effectively suppress pain sensation, but most of these compounds act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable molecule, quaternary ammonium–azobenzene–quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRTRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain.
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Immuno- and Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity
17.02.2012
Cell,
2012,
DOI 10.1016/j.cell.2011.12.030,
Volume 148, Issue 4, 727-738,
published on 17.02.2012
Cell, online article
Cell, online article
Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit β5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of β5c/X but not β5i, thereby explaining the selectivity of PR-957 for β5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.
Discovery and Mutagenicity of a Guanidinoformimine Lesion as a new Intermediate of the Oxidative Deoxyguanosine Degradation Pathway
13.02.2012
Jacs,
2012,
DOI: 10.1021/ja211435d,
134 (10), pp 4925–4930
published on 13.02.2012
Oxidative degradation of DNA is a major mutagenic process. Reactive oxygen species (ROS) produced in the course of oxidative phosphorylation or by exogenous factors are known to attack preferentially deoxyguanosine. The latter decomposes to give mutagenic lesions, which under physiological conditions are efficiently repaired by specialized maintenance systems in the cell. Although many intermediates of the degradation pathway are today well-known, we report in this study the discovery of a new intermediate with an interesting guanidinoformimine structure. The structure elucidation of the new lesion was possible by using HPLC–MS techniques and organic synthesis. Finally we report the mutagenic potential of the new lesion in comparison to the known lesions imidazolone and oxazolone using primer extension and pyrosequencing experiments.
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Consecutive Proteolytic Digestion in an Enzyme Reactor Increases Depth of Proteomic and Phosphoproteomic Analysis
12.02.2012
Anal. Chem.,
2012,
DOI: 10.1021/ac300006b,
84 (6), pp 2631–2637
published on 12.02.2012
Analytical advantages of using multiple enzymes for sample digestion (MED), primarily an increase of sequence coverage, have been reported in several studies. However, this approach is only rarely used, mainly because it requires additional sample and mass spectrometric measurement time. We have previously described Filter Aided Sample Preparation (FASP), a type of proteomic reactor, in which samples dissolved in sodium dodecyl sulfate (SDS) are digested in an ultrafiltration unit. In FASP, such as in any other preparation protocol, a portion of sample remains after digestion and peptide elution. Making use of this fact, we here develop a protocol enabling consecutive digestion of the sample with two or three enzymes. By use of the FASP method, peptides are liberated after each digestion step and remaining material is subsequently cleaved with the next proteinase. We observed excellent performance of the ultrafiltration devices in this mode, allowing efficient separation of orthogonal populations of peptides, resulting in an increase in the numbers of identified peptides and proteins. At the low microgram level, we found that the consecutive use of endoproteinases LysC and trypsin enabled identification of up to 40% more proteins and phosphorylation sites in comparison to the commonly used one-step tryptic digestion. MED-FASP offers efficient exploration of previously unused sample material, increasing depth of proteomic analyses and sequence coverage.
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Crystal Structure of Methylornithine Synthase (PylB): Insights into the Pyrrolysine Biosynthesis
07.02.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201106765,
51, 1339 –1342,
published on 07.02.2012
Angewandte Chemie, online article
Angewandte Chemie, online article
Pyrrolysine is the 22nd amino acid that is encoded by the natural genetic code. In the archaebacterial family Methanosarcinaceae, its incorporation into three proteins (MtmB, MtbB, and MttB) involved in the methylamine catabolic pathway is specified by the amber stop codon UAG. The unusual amino acid was discovered in 2002 by crystallography and mass spectrometry of MtmB, MtbB, and MttB. Asophisticated bioinformatics strategy has shown a set of five genes (pylBCDST) to be both
necessary and sufficient for the biosynthesis and utilization of Pyrrolysin. More specifically, pylT and pylS code for a pyrrolysine tRNA and its cognate amino acyl tRNA synthetase, whereas three enzymes specified by pylB, pylC, and pylD catalyze pyrrolysine biosynthesis. Incorporation experiments using lysine and recombinant Escherichia coli strains engineered for expression of mtmB and pylBCDST genes of Methanosarcina barkeri, showed that all the carbon and nitrogen atoms of Pyrrolysine are derived from lysine.
Exploiting nature’s rich source of proteasome inhibitors as starting points in drug development
06.02.2012
Chem. Commun.,
2012,
DOI: 10.1039/c1cc15273d,
2012, 48, 1364–1378
published on 06.02.2012
Chem. Commun., online article
Chem. Commun., online article
Cancer is the No. 2 cause of death in the Western world and one of the most expensive diseases to treat. Thus, it is not surprising, that every major pharmaceutical and biotechnology company has a blockbuster oncology product. In 2003, Millennium Pharmaceuticals entered the race with Velcades, a first-in-class proteasome inhibitor that has been approved by the FDA for treatment of multiple
myeloma and its sales have passed the billion dollar mark. Velcades’s extremely toxic boronic acid pharmacophore, however, contributes to a number of severe side effects. Nevertheless, the launching of this product has validated the proteasome as a target in fighting cancer and further proteasome inhibitors have entered the market as anti-cancer drugs. Additionally, proteasome inhibitors have found application as crop protection agents, anti-parasitics, immunosuppressives, as well as in new therapies
for muscular dystrophies and inflammation. Many of these compounds are based on microbial metabolites. In this review, we emphasize the important role of the structural elucidation of the various unique binding mechanisms of these compounds that have been optimized throughout evolution to target the proteasome. Based on this knowledge, medicinal chemists have further optimized these natural products, resulting in potential drugs with reduced off-target activities.
Crystal Structures of Mutant IspH Proteins Reveal a Rotation of the Substrate's Hydroxymethyl Group during Catalysis
06.02.2012
Journal of Molecular Biology,
2012,
doi:10.1016/j.jmb.2011.11.033,
Volume 416, pages 1–9,
published on 06.02.2012
Journal of Molecular Biology, online article
Journal of Molecular Biology, online article
Isoprenoids derive from two universal precursors, isopentenyl diphosphate and dimethylallyl diphosphate, which in most human pathogens are synthesized in the deoxyxylulose phosphate pathway. The last step of
this pathway is the conversion of (E)-1-hydroxy-methylbut-2-enyl-4-diphosphate into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate catalyzed by the iron–sulfur protein IspH. The crystal structures
reported here of the IspHmutant proteins T167C, E126Dand E126Qreveal an alternative substrate conformation compared to the wild-type structure. Thus, the previously observed alkoxide complex decomposes, and the substrate's hydroxymethyl group rotates to interact with Glu126. The carboxyl group of Glu126 then donates a proton to the hydroxyl group to enable water elimination. The structural and functional studies provide further knowledge of the IspH reaction mechanism, which opens up new routes to inhibitor design against malaria and tuberculosis.
An Approach to Aminonaphthoquinone Ansamycins Using a Modified Danishefsky Diene
01.02.2012
Org. Letters,
2012,
DOI: 10.1021/ol203437a,
published on 01.02.2012
A robust and scalable synthesis of a novel, cyano-substituted Danishefsky-type diene and its use in the Diels-Alder reaction with various dienophiles is reported. The diene allows for the rapid construction of highly substituted aminonaphthoquinones that occur in numerous ansamycin antibiotics.
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Insights into the Structural Network Responsible for Oligomerization and Activity of the Bacterial Virulence Regulator Caseinolytic Protease P (Clpp)
30.01.2012
Journal of Biological Chemistry,
2012,
doi:10.1074/jbc.M111.336222,
published on 30.01.2012
Journal of Biological Chemistry, online article
Journal of Biological Chemistry, online article
The barrel-shaped ClpP protease is a main virulence regulator in the bacterial pathogen Staphylococcus aureus. It consists of two heptameric rings forming a omo-tetradecamer with an inner chamber that houses the 14 active sites. We recently showed that SaClpP3 is able
to adopt a compressed, inactive conformation. We here present the 2.3 Å resolution structure of SaClpP in its closed, active conformation as well as the structure of the S98A mutant. Comprehensive mutational analysis aiming at
destabilizing one or the other or both conformations was able to pinpoint key residues involved in this catalytic switch and in the heptamer-heptamer interaction. By probing the active site serine with a covalently modifying
beta-lacton probe, we could show that the tetradecameric organization is essential for a proper formation of the active site. Structural data suggest that a highly conserved hydrogenbonding network links oligomerization to
activity. A comparison of ClpP structures from different organisms provides suggestive evidence for the presence of a universal mechanism regulating ClpP protease activity in which binding of one subunit to the corresponding subunit on the other ring interface is necessary for the functional assembly of the catalytic triad and thus for
protease function. This mechanism ensures controlled access to the active sites of a highly unspecific protease.
Low values of 5-hydroxymethylcytosine (5hmC), the ‘‘sixth base,’’ are associated with anaplasia in human brain tumors
10.01.2012
Int. J. Cancer,
2012,
DOI: 10.1002/ijc.27429,
131, 1577–1590
published on 10.01.2012
5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5 mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5 hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC measurements were unrelated to 5 mC values. We find that the number of 5 hmC positive cells and the amount of 5 hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.
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Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors
10.01.2012
Nature Chemistry,
2012,
doi:10.1038/nchem.1234,
4, 105–111
published on 10.01.2012
Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with lightsensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric alpha3beta4 and alpha4beta2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.
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Mechanism of UV-Induced Formation of Dewar Lesions in DNA
09.01.2012
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201106231,
Volume 51, Issue 2, pages 408–411,
published on 09.01.2012
Angewandte Chemie, online article
Angewandte Chemie, online article
Living organisms exposed to sunlight are constantly challenged
by the formation of UV-induced lesions in DNA, which induce cell death and cause mutations. UVirradiation of TpT and TpC sequences leads to the formation of two primary lesions, namely cyclobutane-pyrimidine dimers (CPD)and lesions, as depicted in Scheme 1. These lesions possess an additional absorption at lmax=320 nm and rearrange to give Dewar valence isomers.
The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop
06.01.2012
The EMBO Journal,
2012,
doi:10.1038/emboj.2011.472,
published on 06.01.2012
The EMBO Journal, online article
The EMBO Journal, online article
Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90- binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous a-helical folds as determined by NMR. These, and especially
DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A– TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal
domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1–DP1 module may serve as an Hsp70–client delivery system for the TPR2A–TPR2B–DP2 segment, which is required for client activation in vivo.
Beta-Lactams and Beta-lactones as activity-based probes in chemical biology
04.01.2012
Med. Chem. Commun.,
2011,
DOI: 10.1039/C2MD00275B,
published on 04.01.2012
Activity-based protein profiling (ABPP) employs small molecule probes to profile their dedicated targets in complex proteomes. Unlike traditional proteomics which is limited on protein abundance,
probes that selectively target the active site of certain proteins are a benign measure of protein activity and provide tools for functional analysis. ABPP probes have largely replaced isotope labelled probes and have demonstrated broad spectrum utility ranging from identification and characterization of disease associated enzymes to drug development. Privileged structures with balanced reactivity are
prime candidates for the design of activity-based probes. beta-Lactams and beta -lactones display such privileged structures and have demonstrated unprecedented value as probes. This review provides an overview on beta -lactam and beta -lactone probes and recent advances in their applications in chemical biology.
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Tetraalkylcuprates(III): Formation, Association, and Intrinsic Reactivity
01.01.2012
Jacs,
2012,
DOI: 10.1021/ol203437a,
2012, 134 (1), pp 613–622
published on 01.01.2012
Tetraalkylcuprates are prototypical examples of organocopper(III) species, which remained elusive until their recent detection by NMR spectroscopy. In agreement with the NMR studies, the present electrospray ionization mass spectrometric experiments, as well as supporting electrical conductivity measurements, indicate that LiCuMe2·LiCN reacts with a series of alkyl halides RX. The resulting Li+Me2CuR(CN)− intermediates then afford the observable Me3CuR– tetraalkylcuprate anions upon Me/CN exchanges with added MeLi. In contrast, the reactions of LiCuMe2·LiCN with neopentyl iodide and various aryl halides give rise to halogen–copper exchanges. Concentration- and solvent-dependent studies suggest that lithium tetraalkylcuprates are not fully dissociated in ethereal solvents, but partly form Li+Me3CuR– contact ion pairs and presumably also triple ions LiMe6Cu2R2–. According to theoretical calculations, these triple ions consist of two square-planar Me3CuR– subunits binding to a central Li+ ion. Upon fragmentation in the gas phase, the mass-selected Me3CuR– anions undergo reductive elimination, yielding both the cross-coupling products MeR and the homocoupling product Me2. The branching between these two fragmentation channels markedly depends on the nature of the alkyl substituent R. The triple ions LiMe6Cu2R2– (as well as their mixed analogues LiMe6Cu2R(R′)−) also afford both cross-coupling and homocoupling products upon fragmentation, but strongly favor the former. On the basis of theoretical calculations, we rationalize this prevalence of cross-coupling by the preferential interaction of the central Li+ ion of the triple ions with two Me groups of each Me3CuR– subunit, which thereby effectively blocks the homocoupling channel. Our results thus show how a Li+ counterion can alter the reactivity of an organocopper species at the molecular level.
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