Yasser A. Elnakady

Complete genome sequence of the myxobacterium Sorangium cellulosum.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strains complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase–like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

GeneTrail—advanced gene set enrichment analysis

We present a comprehensive and efficient gene set analysis tool, called ‘GeneTrail’ that offers a rich functionality and is easy to use. Our web-based application facilitates the statistical evaluation of high-throughput genomic or proteomic data sets with respect to enrichment of functional categories. GeneTrail covers a wide variety of biological categories and pathways, among others KEGG, TRANSPATH, TRANSFAC, and GO. Our web server provides two common statistical approaches, ‘Over-Representation Analysis’ (ORA) comparing a reference set of genes to a test set, and ‘Gene Set Enrichment Analysis’ (GSEA) scoring sorted lists of genes. Besides other newly developed features, GeneTrails statistics module includes a novel dynamic-programming algorithm that improves the P-value computation of GSEA methods considerably. GeneTrail is freely accessible at http://genetrail.bioinf.uni-sb.de

Mechanism of action of tubulysin, an antimitotic peptide from myxobacteria.

Tubulysin A is a highly cytotoxic peptide with antimitotic activity that induces depletion of cell microtubules and triggers the apoptotic process. Treated cells accumulated in the G2/M phase. Tubulysin A inhibited tubulin polymerization more efficiently than vinblastine and induced depolymerization of isolated microtubule preparations. Microtubule depolymerization could not be prevented by preincubation with epothilone B and paclitaxel, neither in cell‐free systems nor in cell lines. In competition experiments, tubulysin A strongly interfered with the binding of vinblastine to tubulin in a noncompetitive way; the apparent Ki was 3 μM. Electron microscopy investigations showed that tubulysin A induced the formation of rings, double rings, and pinwheel structures. The mode of action of tubulysin A resembled that of peptide antimitotics dolastatin 10, phomopsin A, and hemiasterlin. Efforts are underway to develop this new group of compounds as anticancer drugs.

Ratjadones inhibit nuclear export by blocking CRM1/exportin 1.

In addition to previously isolated ratjadone A we describe three new members of this family, ratjadones B, C, and D, from another strain of the myxobacterium Sorangium cellulosum. We have investigated the properties of these ratjadones with respect to their activity on mammalian cell lines. We found IC(50) values in the picomolar range and a significant increase in the size of nuclei. A further examination showed that they inhibit the export of the leucine-rich nuclear export signal (LR-NES) containing proteins in different cell lines. Ratjadones are able to inhibit the formation of the nuclear export complex composed of the CRM1, RanGTP, and the cargo protein, as shown by two different in vitro assays. Finally, the binding of ratjadone C to CRM1 was demonstrated. These ratjadone activities are in the same concentration range as described for the polyketide leptomycin B (LMB) from Streptomyces sp. Like LMB, it seems that the ratjadones covalently bind to CRM1, inhibit cargo protein binding via LR-NES, and thereby block nuclear export. Thus, the ratjadones represent a new class of natural compounds which inhibit proliferation in eukaryotes by blocking nuclear export.

Pretubulysin, a Potent and Chemically Accessible Tubulysin Precursor from Angiococcus disciformis

Simplify, simplify, simplify! Pretubulysin (structure without the green substituents), a simplified tubulysin was prepared in the laboratory and also found in a natural myxobacterial source. This biosynthetic precursor of the tubulysins is not as active as tubulysins A and D but is still effective in picomolar concentrations against cancer cell lines.

Mutasynthesis-derived myxalamids and origin of the isobutyryl-CoA starter unit of myxalamid B.

Myxalamids are potent inhibitors of the eukaryotic electron transport chain produced by different myxobacteria. Here, we describe the identification of the myxalamid biosynthesis gene cluster from Myxococcus xanthus. Additionally, new myxalamids (5–13) have been obtained by mutasynthesis from bkd mutants of M. xanthus and Stigmatella aurantiaca. Moreover, as these bkd mutants are still able to produce myxalamid B (2), the origin of the isobutyryl‐CoA (IB‐CoA) starter unit required for its biosynthesis has been determined. In a M. xanthus bkd mutant, IB‐CoA originates from valine, but in S. aurantiaca this starter unit is derived from α‐oxidation of iso‐odd fatty acids, thereby connecting primary and secondary metabolism.

Pretubulysin: From Hypothetical Biosynthetic Intermediate to Potential Lead in Tumor Therapy

Pretubulysin is a natural product that is found in strains of myxobacteria in only minute amounts. It represents the first enzyme-free intermediate in the biosynthesis of tubulysins and undergoes post-assembly acylation and oxidation reactions. Pretubulysin inhibits the growth of cultured mammalian cells, as do tubulysins, which are already in advanced preclinical development as anticancer and antiangiogenic agents. The mechanism of action of this highly potent compound class involves the depolymerization of microtubules, thereby inducing mitotic arrest. Supply issues with naturally occurring derivatives can now be circumvented by the total synthesis of pretubulysin, which, in contrast to tubulysin, is synthetically accessible in gram-scale quantities. We show that the simplified precursor is nearly equally potent to the parent compound. Pretubulysin induces apoptosis and inhibits cancer cell migration and tubulin assembly in vitro. Consequently, pretubulysin appears to be an ideal candidate for future development in preclinical trials and is a very promising early lead structure in cancer therapy.

Evidence for the Mode of Action of the Highly Cytotoxic Streptomyces Polyketide Kendomycin

The macrocyclic polyketide kendomycin exhibits antiosteoporotic and antibacterial activity, as well as strong cytotoxicity against multiple human tumor cell lines. Despite the promise of this compound in several therapeutic areas, the cellular target(s) of kendomycin have not been identified to date. We have used a number of approaches, including microscopy, proteomics, and bioinformatics, to investigate the mode of action of kendomycin in mammalian cell cultures. In response to kendomycin treatment, human U‐937 tumor cells exhibit depolarization of the mitochondrial membrane, caspase 3 activation, and DNA laddering, consistent with induction of the intrinsic apoptotic pathway. To elucidate possible apoptotic triggers, DIGE and MALDI‐TOF were used to identify proteins that are differently regulated in U‐937 cells relative to controls. Statistical analysis of the proteomics data by the new web‐based application GeneTrail highlighted several significant changes in protein expression, most notably among proteasomal regulatory subunits. Overall, the profile of altered expression closely matches that observed with other tumor cell lines in response to proteasome inhibition. Direct assay in vitro further shows that kendomycin inhibits the chymotrypsin‐like activity of the rabbit reticulocyte proteasome, with comparable efficacy to the established inhibitor MG‐132. We have also demonstrated that ubiquitinylated proteins accumulate in kendomycin‐treated U‐937 cells, while vacuolization of the endoplasmic reticulum and mitochondrial swelling are induced in a second cell line derived from kangaroo rat epithelial (PtK2) cells, phenotypes classically associated with inhibition of the proteasome. This study therefore provides evidence that kendomycin mediates its cytotoxic effects, at least in part, through proteasome inhibition.

Preparative mass-spectrometry profiling of bioactive metabolites in Saudi-Arabian propolis fractionated by high-speed countercurrent chromatography and off-line atmospheric pressure chemical ionization mass-spectrometry injection

Propolis is a glue material collected by honeybees which is used to seal cracks in beehives and to protect the bee population from infections. Propolis resins have a long history in medicinal use as a natural remedy. The multiple biological properties are related to variations in their chemical compositions. Geographical settings and availability of plant sources are important factors for the occurrence of specific natural products in propolis. A propolis ethylacetate extract (800mg) from Saudi Arabia (Al-Baha region) was separated by preparative scale high-speed countercurrent chromatography (HSCCC) using a non-aqueous solvent system n-hexane-ACN (1:1, v/v). For multiple metabolite detection, the resulting HSCCC-fractions were sequentially injected off-line into an atmospheric pressure chemical ionization mass-spectrometry (APCI-MS/MS) device, and a reconstituted mass spectrometry profile of the preparative run was visualized by selected ion traces. Best ion-intensities for detected compounds were obtained in the negative APCI mode and monitored occurring co-elution effects. HSCCC and successive purification steps resulted in the isolation and characterization of various bioactive natural products such as (12E)- and (12Z)-communic acid, sandaracopimaric acid, (+)-ferruginol, (+)-totarol, and 3β-acetoxy-19(29)-taraxasten-20a-ol using EI-, APCI-MS and 1D/2D-NMR. Cycloartenol-derivatives and triterpene acetates were isolated in mixtures and elucidated by EI-MS and 1D-NMR. Free fatty acids, and two labdane fatty acid esters were identified by APCI-MS/MS. In total 19 metabolites have been identified. The novel combination of HSCCC fractionation, and APCI-MS-target-guided molecular mass profiling improve efficiency of lead-structure identification.

Proteome analysis of Sorangium cellulosum employing 2D-HPLC-MS/MS and improved database searching strategies for CID and ETD fragment spectra.

Shotgun proteome analysis of the myxobacterial model strain for secondary metabolite biosynthesis Sorangium cellulosum was performed employing off-line two-dimensional high-pH reversed-phase HPLC x low-pH ion-pair reversed-phase HPLC and dual tandem mass spectrometry with collision-induced dissociation (CID) and electron transfer dissociation (ETD) as complementary fragmentation techniques. Peptide identification using database searching was optimized for ETD fragment spectra to obtain the maximum number of identifications at equivalent false discovery rates (1.0%) in the evaluation of both fragmentation techniques. In the database search of the CID MS/MS data, the mass tolerance was set to the well-established 0.3 Da window, whereas for ETD data, it was widened to 1.1 Da to account for hydrogen-rearrangement in the radical-intermediate of the peptide precursor ion. To achieve a false discovery rate comparable to the CID results, we increased the significance threshold for peptide identification to 0.001 for the ETD data. The ETD based analysis yielded about 74% of all peptides and about 78% of all proteins compared to the CID-method. In the combined data set, 952 proteins of S. cellulosum were confidently identified by at least two peptides per protein, facilitating the study of the function of regulatory proteins in the social myxobacteria and their role in secondary metabolism.