Pretubulysin: a new option for the treatment of metastatic cancer

Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-x_L, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCF^Fbw7 E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer.     

The Diversity and Evolution of Wolbachia Ankyrin Repeat Domain Genes

Ankyrin repeat domain-encoding genes are common in the eukaryotic and viral domains of life, but they are rare in bacteria, the exception being a few obligate or facultative intracellular Proteobacteria species. Despite having a reduced genome, the arthropod strains of the alphaproteobacterium Wolbachia contain an unusually high number of ankyrin repeat domain-encoding genes ranging from 23 in wMel to 60 in wPip strain. This group of genes has attracted considerable attention for their astonishing large number as well as for the fact that ankyrin proteins are known to participate in protein-protein interactions, suggesting that they play a critical role in the molecular mechanism that determines host-Wolbachia symbiotic interactions. We present a comparative evolutionary analysis of the wMel-related ankyrin repeat domain-encoding genes present in different Drosophila-Wolbachia associations. Our results show that the ankyrin repeat domain-encoding genes change in size by expansion and contraction mediated by short directly repeated sequences. We provide examples of intra-genic recombination events and show that these genes are likely to be horizontally transferred between strains with the aid of bacteriophages. These results confirm previous findings that the Wolbachia genomes are evolutionary mosaics and illustrate the potential that these bacteria have to generate diversity in proteins potentially involved in the symbiotic interactions.     

Combination of a Proteomics Approach and Reengineering of Meso Scale Network Models for Prediction of Mode-of-Action for Tyrosine Kinase Inhibitors

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Thyroid hormone receptor α1 is a critical regulator for the expression of ion channels during final differentiation of outer hair cells

Cochlear outer hair cells (OHCs) terminally differentiate prior to the onset of hearing. During this time period, thyroid hormone (TH) dramatically influences inner ear development. It has been shown recently that TH enhances the expression of the motor protein prestin via liganded TH receptor β (TRβ) while in contrast the expression of the potassium channel KCNQ4 is repressed by unliganded TRα1. These different mechanisms of TH regulation by TRα1 or TRβ prompted us to analyse other ion channels that are required for the final differentiation of OHCs. We analysed the onset of expression of the Ca²⁺ channel Ca_V1.3, and the K⁺ channels SK2 and BK and correlated the results with the regulation via TRα1 or TRβ. The data support the hypothesis that proteins expressed in rodents prior to or briefly after birth like Ca_V1.3 and prestin are either independent of TH (e.g. Ca_V1.3) or enhanced through TRβ (e.g. prestin). In contrast, proteins expressed in rodents later than P6 like KCNQ4 (∼P6), SK2 (∼P9) and BK (∼P11) are repressed through TRα1. We hypothesise that the precise regulation of expression of the latter genes requires a critical local TH level to overcome the TRα1 repression. Harald Winter and Claudia Braig contributed equally to this work.     

Genetic and environmental heterogeneity of residual variance of weight traits in Nellore beef cattle

Background

Many studies have provided evidence of the existence of genetic heterogeneity of environmental variance, suggesting that it could be exploited to improve robustness and uniformity of livestock by selection. However, little is known about the perspectives of such a selection strategy in beef cattle.

Methods

A two-step approach was applied to study the genetic heterogeneity of residual variance of weight gain from birth to weaning and long-yearling weight in a Nellore beef cattle population. First, an animal model was fitted to the data and second, the influence of additive and environmental effects on the residual variance of these traits was investigated with different models, in which the log squared estimated residuals for each phenotypic record were analyzed using the restricted maximum likelihood method. Monte Carlo simulation was performed to assess the reliability of variance component estimates from the second step and the accuracy of estimated breeding values for residual variation.

Results

The results suggest that both genetic and environmental factors have an effect on the residual variance of weight gain from birth to weaning and long-yearling in Nellore beef cattle and that uniformity of these traits could be improved by selecting for lower residual variance, when considering a large amount of information to predict genetic merit for this criterion. Simulations suggested that using the two-step approach would lead to biased estimates of variance components, such that more adequate methods are needed to study the genetic heterogeneity of residual variance in beef cattle.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.     

Protein-protein-interaction Network Organization of the Hypusine Modification System

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: “bioreactor-TAP-MS/MS.” By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.Cellular homeostasis is controlled by signaling networks that communicate through post-translational modifications (PTM)¹ of proteins, including phosphorylation, acetylation and methylation (1–3). These modifications are typically attached to various types of proteins by multiple independent enzymes, and thereby simultaneously regulate a wide range of protein functions. Consequently, most signaling pathways are highly redundant, enabling maintenance of cellular integrity even if the modification of a single signaling molecule is disrupted (4). A striking exception is hypusine. This essential PTM is limited to a single protein: the eukaryotic initiation factor 5A (eIF-5A) (5). Disruption of this PTM leads to growth arrest in proliferating eukaryotic cells and is fatal for the developing mammalian embryo (6, 7). During hypusine biosynthesis, the lysine residue at position 50 (Lys₅₀) in eIF-5A is converted into the unusual amino acid hypusine (N^ε-(4-amino-2-hydroxybutyl)lysine; depicted in Fig. 1A) (5). This process activates eIF-5A and is mediated by two enzymatic reactions: first, deoxyhypusine synthase (DHS) catalyzes the transfer of the 4-aminobutyl moiety of spermidine to the ε-amino group of Lys₅₀ to form an intermediate residue, deoxyhypusine (Dhp₅₀) (8). Subsequently, deoxyhypusine hydroxylase (DOHH) mediates the formation of hypusine (Hyp₅₀) by addition of a hydroxyl group to the deoxyhypusine residue (9). eIF-5A, DHS and DOHH are all essential for proliferation of higher eukaryotic cells (10, 11), and eIF-5A is strictly conserved throughout eukaryotic evolution (12).Open in a separate windowFig. 1.The hypusine modification and TAP fusion proteins employed in this study. A, The hypusine modification pathway and major proposed eIF-5A functions. B, Structure of the plasmid inserts coding for SG-tagged bait proteins. The amino acid positions of eIF-5A mutants are indicated in italic. SBP, streptavidin binding peptide. C, Metabolic incorporation of ³H-labeled spermidine into eIF-5A. Arrowheads indicate bands of SG-tagged and endogenous eIF-5A proteins. D, Anti-Myc-tag Western blot of cell lysates from retrovirally transduced Ba/F3 p210 cell lines for the quantification of constitutively expressed SG-tagged bait proteins. E, Representative TAP outputs for MS/MS analysis, after 1D PAGE separation and Coomassie staining. Separation distance varies from ∼2 to 4 cm.The eIF-5A protein has been proposed to promote various different cellular processes that potentially regulate proliferation, including translation initiation (13) and elongation (14) as well as nucleocytoplasmic transport of RNA or other cargoes (15, 16). Using inhibitors of DHS and DOHH or eIF-5A mutants deficient for hypusine modification, it has also been shown that this modification is a prerequisite of at least a subset of known eIF-5A functions (10, 11, 17, 18). The eIF-5A protein has also been implicated in numerous pathologic conditions including various types of cancer (19–23), β-cell inflammation (and therefore diabetes) (24) and HIV-1 infection (25). Human and rodent cells carry two highly homologous eIF-5A genes coding for distinct isoforms. Although eIF-5A1 is expressed at high levels throughout all tissues, eIF-5A2 is detectable only in a few embryonic tissues as well as adult testis, central nervous system (26), and cancer tissue (21, 22, 27–29).Although there have been ample reports suggesting eIF-5A is involved in translational control, the molecular mechanisms through which it ultimately influences cellular physiology and leads to disease remain unclear. Moreover, it remains equally possible that at least some of eIF-5A''s effects on cellular functions might not involve direct effects on translation. Also, there is no information available on whether the two isoforms of mammalian eIF-5A are functionally congruent.To address these fundamental questions systematically and comprehensively, we employed a bioreactor-based tandem affinity purification (TAP) approach followed by MS identification of purified protein complexes (“bioreactor-TAP-MS/MS”). To obtain a complete interaction map of the proteins involved in hypusine modification, we used this approach to identify interaction partners of both isoforms of eIF-5A, as well as the hypusine modification enzymes DHS and DOHH. In total, we identified 248 proteins that either directly interact with these bait proteins or are components of higher complexes containing the aforementioned proteins. Furthermore, we validated a subset of putative interaction partners of both eIF-5A isoforms, using Western blots of reciprocal TAP experiments, as well as a live-cell protein-fragment complementation assay (PCA). Our analysis provides a molecular framework for a detailed understanding on how this signal transduction pathway affects different crucial cellular functions.     

Expression of Eukaryotic Initiation Factor 5A and Hypusine Forming Enzymes in Glioblastoma Patient Samples: Implications for New Targeted Therapies

Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.