Content
2007
Selective Detection of 5-Methylcytosine Sites in DNA
26.11.2007
After the complete sequencing of the human genome, one of
the next important steps is to decipher the genetic mechanisms
which allow cells to switch genes on and off. These
processes are fundamental for cellular development. At the
chemical level, gene silencing requires the selective methylation
of of the C5-position of cytidines in DNA, and this
established method is today known as the epigenetic control
of gene function. Failure of the epigenetic control of genes
has been connected to many human diseases, including
neurodevelopmental disorders and cancer. The identification
of 5-methylcytosine residues (5MedC) in genes is consequently
of paramount importance for the diagnosis of
modern diseases.
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Secondary structure and distribution of fusogenic LV-peptides in lipid membranes
24.11.2007
European Biophysics Journal,
2007,
37, 4,
435 - 45
published on 24.11.2007
European Biophysics Journal, online article
European Biophysics Journal, online article
LV-peptides were designed as membrane-spanning low-complexity model structures that mimic fusion protein transmembrane domains. These peptides harbor a hydrophobic core sequence that consists of helix-promoting and helix-destabilizing residues at different ratios. Previously, the fusogenicity of these peptides has been shown to increase with the conformational flexibility of their hydrophobic cores as determined in isotropic solution. Here, we examined the secondary structure, orientation, and distribution of LV-peptides in membranes. Our results reveal that the peptides are homogeneously distributed within the membranes of giant unilamellar liposomes and capable of fusing them. Increasing the valine content of the core up to the level of the β-branched residue content of SNARE TMDs (∼50%) enhances fusogenicity while maintaining a largely α-helical structure in liposomal membranes. A further increase in valine content or introduction of a glycine/proline pair favors β-sheet formation. In planar bilayers, the α-helices adopt oblique angles relative to the bilayer normal and the ratio of α-helix to β-sheet responds more sensitively to valine content. We propose that the fusogenic conformation of LV-peptides is likely to correspond to a membrane-spanning α-helix. β-Sheet formation in membranes may be considered a side-reaction whose extent reflects conformational flexibility of the core.
CIPSM-Speaker Thomas Carell wins 2008 Otto-Bayer-Preis
23.11.2007
CIPSM-Researcher and Speaker Thomas Carell wins the prestigious Otto-Bayer-Preis 2008 for his work of the understanding of light-triggered DNA-lesions.
Thomas is a specialist for the synthesis of DNA-lesions and lesion analogues, and their incorporation into DNA-strands and the investigations of the resulting structures (x-ray, enzymatic digestion) and biological fate (repair experiments, mutagenicity) of the damaged oligonucleotides.
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Press commentaries:
Informationsdienst Wissenschaft
Sueddeutsche Zeitung, November 28th 2007 / S. 54:
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Nature Spotlight on Germany
22.11.2007
Nature,
2007,
Nature Jobs,
452-3 and 485
published on 19.11.2007
www.nature.com, free online article
www.nature.com, free online article
Seeing Cisplatin - Discerning how an anticancer drug meddles with replication, transcription
12.11.2007
Chemical & Engineering News,
2007,
Vol. 85, Nr. 46,
13
published on 12.11.2007
Chemical & Engineering News, online article
Chemical & Engineering News, online article
CISPLATIN HAS BEEN USED to treat cancer for decades. But the drug's detailed molecular interactions with the enzymes it aims to interrupts have remained obscure. In two separate papers, researchers are now reporting the first crystal structures of cisplatin complexed with two key enzymes.
"For the first time, we are getting deep insight on what goes on at the atomic level with cisplatin and cellular machinery," says Thomas Carell, a protein chemist at the University of Munich.
Cisplatin creates bulky platinum adducts with pairs of guanidine bases that are side-by-side or one base apart on genomic DNA. The extra bulk blocks most DNA polymerases that slide along DNA when they copy the genetic code during cell division. The adducts also obstruct RNA polymerases, which transcribe DNA to make proteins. But no structures of cisplatin in complex with either DNA or RNA polymerases have come to light until now.
In one report, Carell and Karl-Peter Hopfner announce the structure of a renegade DNA polymerase called Pol η. Normal DNA polymerases get stalled by cisplatin because their grip on DNA is so tight that there's no room for adduct riffraff. But Pol η manages to replicate over unwelcome DNA adducts.
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Original Science article, Science 2007, 318, 967
See also under www.cipsm.de
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Mechanism of transcriptional stalling at cisplatindamaged DNA
11.11.2007
Nature Structural & Molecular Biology,
2007,
doi:10.1038/nsmb1314,
1-7
published on 11.11.2007
www.nature.com/nsmb, online article
www.nature.com/nsmb, online article
The anticancer drug cisplatin forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall RNA polymerase II (Pol II)
and trigger transcription-coupled DNA repair. Here we present a structure-function analysis of Pol II stalling at a cisplatin lesion
in the DNA template. Pol II stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP
misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according
to an ‘A-rule’ known for DNA polymerases. Pol II can bypass a cisplatin lesion that is artificially placed beyond the translocation
barrier, even in the presence of a G.A mismatch. Thus, the barrier prevents transcriptional mutagenesis. The stalling mechanism
differs from that of Pol II stalling at a photolesion, which involves delivery of the lesion to the active site and lesion-templated
misincorporation that blocks transcription.
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Bypass of DNA Lesions Generated During Anticancer Treatment with Cisplatin by DNA Polymerase Eta
09.11.2007
DNA polymerase eta (Pol eta) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UVinduced cis–syn thymine dimers. We present structural and biochemical analysis of how Pol h copies Pt-GG–containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol eta to replicate across strongly distorting DNA lesions.
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Press commentaries:
Lübecker Nachrichten www.LN-online.de
Other articles can be found under www.handelsbaltt.de, www.Pharmazeutische-Zeitung.de and Frankfurter Neue Presse by searching for Carell or Hopfner
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In Vivo Chemoenzymatic Control of N-Terminal Processing in Recombinant Human Epidermal Growth Factor
08.11.2007
Protein synthesis initiates with Met in the cytosol of eukaryotes
and formylmethionine (fMet) in prokaryotes and eukaryotic organelles. N-terminal methionine excision (NME) is the major
source of N-terminal amino acid diversity in all three life kingdoms. The excision is dictated by the nature and bulkiness of the side-chain of the second amino acid (the penultimate residue) and is catalyzed by methionylaminopeptidases (MetAPs; EC 3.4.11.18). In bacteria, Lys, Arg, Leu, Phe and Ile protect the N-terminal Met from removal, whereas Met excision is promoted by having Gly, Ala, Pro, Cys, Ser, Thr or Val as the penultimate residues. NME is an irreversible cotranslational proteolysis, and is completed before the nascent polypeptide chains are fully synthesized. It was recently estimated
that between 55 and 70% of the proteins of any given proteome undergo this Met excision.
Structural Insight into the Dual Ligand Specificity and Mode of High Density Lipoprotein Association of Apolipoprotein D
19.10.2007
Human apolipoproteinD(ApoD) occurs in plasma associated
with high density lipoprotein. Apart from the involvement in
lipid metabolism, its binding activity for progesterone and
arachidonic acid plays a role in cancer development and neurological
diseases. The crystal structures of freeApoDand its complex
with progesterone were determined at 1.8A˚ resolution and
reveal a lipocalin fold. The narrow, mainly uncharged pocket
within the typical beta-barrel accommodates progesterone with its
acetyl side chain oriented toward the bottom. The cavity adopts
essentially the same shape in the absence of progesterone and
allows complexation of arachidonic acid as another cognate
ligand. Three of the four extended loops at the open end of the
beta-barrel expose hydrophobic side chains, which is an unusual
feature for lipocalins and probably effects association with the
high density lipoprotein particle by mediating insertion into the
lipid phase. This mechanism is in line with an unpaired Cys
residue in the same surface region that can form a disulfide
cross-link with apolipoprotein A-II.
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miRNAs Get an Early Start on Translational Silencing
05.10.2007
Cell,
2007,
131,
25-8
published on 05.10.2007
www.cell.com, online article
www.cell.com, online article
MicroRNAs (miRNAs) control gene expression by regulating mRNA stability and translation.
Using cell-free in vitro systems, several labs have recently reported insights into the molecular mechanisms underlying miRNA-guided translational repression (Kiriakidou et al., 2007; Mathonnet et al., 2007; Thermann and Hentze, 2007; Wakiyama et al., 2007). These
new findings indicate that miRNAs inhibit translation at early steps of initiation.
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Phenylalanine Promotes Interaction of Transmembrane Domains via GxxxG Motifs
26.09.2007
Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/ POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the −3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane
integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.
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The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins
14.06.2007
Nature Protocols,
2007,
Vol. 2 No. 6,
1528-35
published on 14.06.2007
www.nature.com/natureprotocols, online article
www.nature.com/natureprotocols, online article
The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity
toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild
purification of corresponding Strep-tag II fusion proteins—including their complexes with interacting partners—both from bacterial
and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be
accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion
proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a
versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.
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CPD Damage Recognition by Transcribing RNA Polymerase II
09.02.2007
Cells use transcription-coupled repair (TCR) to efficiently eliminate DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs). Here we present the structure-based mechanism for the first step in eukaryotic TCR, CPD-induced stalling of RNA polymerase (Pol) II. A CPD in the transcribed strand slowly passes a translocation barrier, and enters the polymerase active site. The CPD 5’-thymine then directs uridine misincorporation into mRNA, which blocks translocation. Artificial replacement of the uridine by adenosine enables CPD bypass, thus Pol II stalling requires CPD-directed misincorporation. In the stalled complex, the lesion is inaccessible, and the polymerase conformation is unchanged. This is consistent with non-allosteric recruitment of repair factors and excision of a lesion-containing DNA fragment in the presence of Pol II.
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