The Mass Distance Fingerprint: a statistical framework for de novo detection of predominant modifications using high-accuracy mass spectrometry

We describe a statistical measure, Mass Distance Fingerprint, for automatic de novo detection of predominant peptide mass distances, i.e., putative protein modifications. The method's focus is to globally detect mass differences, not to assign peptide sequences or modifications to individual spectra. The Mass Distance Fingerprint is calculated from high accuracy measured peptide masses. For the data sets used in this study, known mass differences are detected at electron mass accuracy or better. The proposed method is novel because it works independently of protein sequence databases and without any prior knowledge about modifications. Both modified and unmodified peptides have to be present in the sample to be detected. The method can be used for automated detection of chemical/post-translational modifications, quality control of experiments and labeling approaches, and to control the modification settings of protein identification tools. The algorithm is implemented as a web application and is distributed as open source software.     

A workflow to increase the detection rate of proteins from unsequenced organisms in high-throughput proteomics experiments

We present and evaluate a strategy for the mass spectrometric identification of proteins from organisms for which no genome sequence information is available that incorporates cross-species information from sequenced organisms. The presented method combines spectrum quality scoring, de novo sequencing and error tolerant BLAST searches and is designed to decrease input data complexity. Spectral quality scoring reduces the number of investigated mass spectra without a loss of information. Stringent quality-based selection and the combination of different de novo sequencing methods substantially increase the catalog of significant peptide alignments. The de novo sequences passing a reliability filter are subsequently submitted to error tolerant BLAST searches and MS-BLAST hits are validated by a sampling technique. With the described workflow, we identified up to 20% more groups of homologous proteins in proteome analyses with organisms whose genome is not sequenced than by state-of-the-art database searches in an Arabidopsis thaliana database. We consider the novel data analysis workflow an excellent screening method to identify those proteins that evade detection in proteomics experiments as a result of database constraints.     

Influence of systematically varied nano-scale topography on cell morphology and adhesion

The types of cell-matrix adhesions and the signals they transduce strongly affect the cell-phenotype. We hypothesized that cells sense and respond to the three-dimensionality of their environment, which could be modulated by nano-structures on silicon surfaces. Human foreskin fibroblasts were cultured on nano-structures with different patterns (nano-post and nano-grate) and heights for 3 days. The presence of integrin alpha(5), beta(1), beta(3), paxillin and phosphorylated FAK (pFAK) were detected by western blot and immunofluorescence. Integrin beta(3) exhibited stronger signals on nano-grates. pFAK and paxillin were observed as small dot-like patterns on the cell-periphery on nano-posts and as elongated and aligned patterns on nano-grates. Collectively, our observations highlighted the presence of focal (integrin beta(1), beta(3), pFAK, paxillin), fibrillar (integrin alpha(5), beta(1)) and 3-D matrix (integrin alpha(5), beta(1), paxillin) adhesions on nano-structures. The presented nano-structures offer interesting opportunities to study the interaction of cells with topographical features comparable to the size of extracellular matrix components.     

MDM2 splice variants predominantly localize to the nucleoplasm mediated by a COOH-terminal nuclear localization signal

Of the >40 alternative and aberrant splice variants of MDM2 that have been described to date, the majority has lost both the well-characterized nuclear localization signal (NLS1) and the nuclear export signal (NES) sequence. Because cellular localization of proteins provides insight regarding their potential function, we determined the localization of three different MDM2 splice variants. The splice variants chosen were the common variants MDM2-A and MDM2-B. In addition, MDM2-FB26 was chosen because it is one of the few variants described that contains the complete p53-binding site. All three splice variants predominantly localized to the nucleus. Nuclear localization of MDM2-A and MDM2-B was controlled by a previously uncharacterized nuclear localization signal (NLS2), whereas nucleoplasmic localization of MDM2-FB26 was mediated by NLS1. p53 and full-length MDM2 colocalized with the splice variants in the nucleus. MDM2-A and MDM2-B both contain a COOH-terminal RING finger domain, and interaction with full-length MDM2 through this domain was confirmed. MDM2-FB26 was the only splice variant evaluated that contained a p53-binding domain; however, interaction between MDM2-FB26 and p53 could not be shown. p14(ARF) did not colocalize with the splice variants and was predominantly expressed within the nucleoli. In summary, nuclear localization signals responsible for the nucleoplasmic distribution of MDM2 splice variants have been characterized. Colocalization and interaction of MDM2-A and MDM2-B with full-length MDM2 in the nucleus have important physiologic consequences, for example, deregulation of p53 activity.     

Identification of liver‐derived bone morphogenetic protein (BMP)‐9 as a potential new candidate for treatment of colorectal cancer

Colorectal cancer (CRC) is a high‐incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)‐9 and to find out whether the application of BMP‐9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP‐target gene ID1, especially in combination with low expression of the BMP‐inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T‐Orgs) and corresponding non‐malignant areas (N‐Orgs) of three patients. The N‐Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP‐9 stimulation of organoids promoted an enrichment of tumour‐suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP‐9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1.In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP‐9 enhances this ratio, even in the presence of noggin. Thus, BMP‐9 is identified as a novel target for the development of improved anti‐cancer therapies of patients with CRC.     

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.     

Post-insemination selection dominates pre-insemination selection in driving rapid evolution of male competitive ability

Sexual reproduction is a complex process that contributes to differences between the sexes and divergence between species. From a male’s perspective, sexual selection can optimize reproductive success by acting on the variance in mating success (pre-insemination selection) as well as the variance in fertilization success (post-insemination selection). The balance between pre- and post-insemination selection has not yet been investigated using a strong hypothesis-testing framework that directly quantifies the effects of post-insemination selection on the evolution of reproductive success. Here we use experimental evolution of a uniquely engineered genetic system that allows sperm production to be turned off and on in obligate male-female populations of Caenorhabditis elegans. We show that enhanced post-insemination competition increases the efficacy of selection and surpasses pre-insemination sexual selection in driving a polygenic response in male reproductive success. We find that after 10 selective events occurring over 30 generations post-insemination selection increased male reproductive success by an average of 5- to 7-fold. Contrary to expectation, enhanced pre-insemination competition hindered selection and slowed the rate of evolution. Furthermore, we found that post-insemination selection resulted in a strong polygenic response at the whole-genome level. Our results demonstrate that post-insemination sexual selection plays a critical role in the rapid optimization of male reproductive fitness. Therefore, explicit consideration should be given to post-insemination dynamics when considering the population effects of sexual selection.     

Browsing herbivores improve the state and functioning of savannas: A model assessment of alternative land‐use strategies

Changing climatic conditions and unsustainable land use are major threats to savannas worldwide. Historically, many African savannas were used intensively for livestock grazing, which contributed to widespread patterns of bush encroachment across savanna systems. To reverse bush encroachment, it has been proposed to change the cattle‐dominated land use to one dominated by comparatively specialized browsers and usually native herbivores. However, the consequences for ecosystem properties and processes remain largely unclear. We used the ecohydrological, spatially explicit model EcoHyD to assess the impacts of two contrasting, herbivore land‐use strategies on a Namibian savanna: grazer‐ versus browser‐dominated herbivore communities. We varied the densities of grazers and browsers and determined the resulting composition and diversity of the plant community, total vegetation cover, soil moisture, and water use by plants. Our results showed that plant types that are less palatable to herbivores were best adapted to grazing or browsing animals in all simulated densities. Also, plant types that had a competitive advantage under limited water availability were among the dominant ones irrespective of land‐use scenario. Overall, the results were in line with our expectations: under high grazer densities, we found heavy bush encroachment and the loss of the perennial grass matrix. Importantly, regardless of the density of browsers, grass cover and plant functional diversity were significantly higher in browsing scenarios. Browsing herbivores increased grass cover, and the higher total cover in turn improved water uptake by plants overall. We concluded that, in contrast to grazing‐dominated land‐use strategies, land‐use strategies dominated by browsing herbivores, even at high herbivore densities, sustain diverse vegetation communities with high cover of perennial grasses, resulting in lower erosion risk and bolstering ecosystem services.