HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.     

Multiple N-methyltransferases for aromatic alkylamines in brain

This report describes our studies of the enzymatic N-methylation of tryptamine and β-phenylethylamine by fractions of homogenized rat brain with either 5-methyltetrahydrofolic acid (5-MTHF) or S-adenosyl-L-methionine (SAM) as the methyl donor. We found the pH optimum between 6.5 and 7.0 in reaction involving 5-MTHF and either substrate. (We confirmed reports that reactions involving SAM have a pH optimum of 7.9 with either substrate). Enzymatic activity in the presence of 5-MTHF was linear with time and protein concentration. Measures of enzymatic activity in the 100,000 x g supernate of whole rat brain homogenerate were enriched 15–25 fold after Sephadex G-100 fractionation of the 40–50% ammonium sulfate precipitate. Dialysis enhanced the enzyme activity in virtually all of the fractions. Blanks with tryptamine and boiled enzyme gave unexpectedly high counts (comparable to those without enzyme), suggesting possible non-enzymatic methylation in addition to enzymatic N-methylation. We controlled for this by using blanks of substrate plus boiled enzyme. Radioactive products were isographic with N-monomethyltryptamine and N,N-dimethyl tryptamine on thin layer chromatograms. In the presence of 5-MTHF there is low enzymatic affinity (Km = approximately 10^?3 M) for either substrate, suggesting that enzymatic N-methylation might occur only when amine concentrations are abnormally high. Assays in the presence of each donor in combination with each substrate suggested the possible existence of multiple N-methyltransferases, with one specific for tryptamine and the others non-specific for aromatic alkylamines.     

CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation

Background  

Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.     

Local erythropoietin and endothelial progenitor cells improve regional cardiac function in acute myocardial infarction

Background

Expanded endothelial progenitor cells (eEPC) improve global left ventricular function in experimental myocardial infarction (MI). Erythropoietin beta (EPO) applied together with eEPC may improve regional myocardial function even further by anti-apoptotic and cardioprotective effects. Aim of this study was to evaluate intramyocardial application of eEPCs and EPO as compared to eEPCs or EPO alone in experimental MI.

Methods and Results

In vitro experiments revealed that EPO dosed-dependently decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA expression in eEPC of proangiogenic and proinflammatory mediators measured by TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion of the left anterior descending coronary artery of nude rats (n = 8-9). After myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel density were enhanced in the border zone of the infarct area. Moreover, apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to transplantation of eEPCs alone. Regional wall motion of the left ventricle was measured using Magnetic Resonance Imaging. After injection of eEPC in the presence of EPO regional wall motion significantly improved as compared to injection of eEPCs or EPO alone.

Conclusion

Intramyocardial transplantation of eEPC in the presence of EPO during experimental MI improves regional wall motion. This was associated with an increased local inflammation, vasculogenesis and survival of the transplanted cells. Local application of EPO in addition to cell therapy may prove beneficial in myocardial remodeling.

     

SIMPLE: A Sequential Immunoperoxidase Labeling and Erasing Method

The ability to simultaneously visualize expression of multiple antigens in cells and tissues can provide powerful insights into cellular and organismal biology. However, standard methods are limited to the use of just two or three simultaneous probes and have not been widely adopted for routine use in paraffin-embedded tissue. We have developed a novel approach called sequential immunoperoxidase labeling and erasing (SIMPLE) that enables the simultaneous visualization of at least five markers within a single tissue section. Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole, combined with a rapid non-destructive method for antibody–antigen dissociation, we demonstrate the ability to erase the results of a single immunohistochemical stain while preserving tissue antigenicity for repeated rounds of labeling. SIMPLE is greatly facilitated by the use of a whole-slide scanner, which can capture the results of each sequential stain without any information loss. (J Histochem Cytochem 57:899–905, 2009)     

MADAM - An open source meta-analysis toolbox for R and Bioconductor

Background  

Meta-analysis is a major theme in biomedical research. In the present paper we introduce a package for R and Bioconductor that provides useful tools for performing this type of work. One idea behind the development of MADAM was that many meta-analysis methods, which are available in R, are not able to use the capacities of parallel computing yet. In this first version, we implemented one meta-analysis method in such a parallel manner. Additionally, we provide tools for combining the results from a set of methods in an ensemble approach. Functionality for visualization of results is also provided.     

Inferring the biogeographic origins of inter‐continental disjunct endemics using a Bayes‐DIVA approach

The arcto‐Tertiary relictual flora is comprised of many genera that occur non‐contiguously in the temperate zones of eastern Asia, Europe, eastern North America, and western North America. Within each distributional area, species are typically endemic and may thus be widely separated from closely related species within the other areas. It is widely accepted that this common pattern of distribution resulted from of the fragmentation of a once more‐continuous arcto‐Tertiary forest. The historical biogeographic events leading to the present‐day disjunction have often been investigated using a phylogenetic approach. Limitations to these previous studies have included phylogenetic uncertainty and uncertainty in ancestral range reconstructions. However, the recently described Bayes‐DIVA method handles both types of uncertainty. Thus, we used Bayes‐DIVA analysis to reconstruct the stem lineage distributions for 185 endemic lineages from 23 disjunct genera representing 17 vascular plant families. In particular, we asked whether endemic lineages within each of the four distributional areas more often evolved from (1) widespread ancestors, (2) ancestors dispersed from other areas, or (3) endemic ancestors. We also considered which of these three biogeographic mechanisms may best explain the origins of arcto‐Tertiary disjunct endemics in the neotropics. Our results show that eastern Asian endemics more often evolved from endemic ancestors compared to endemics in Europe and eastern and western North America. Present‐day endemic lineages in the latter areas more often arose from widespread ancestors. Our results also provide anecdotal evidence for the importance of dispersal in the biogeographic origins of arcto‐Tertiary species endemic in the neotropics.     

Dissecting Protein Function: An Efficient Protocol for Identifying Separation-of-Function Mutations That Encode Structurally Stable Proteins

Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3⁻ mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.     

Assessment of flexible backbone protein design methods for sequence library prediction in the therapeutic antibody Herceptin-HER2 interface

Computational protein design methods can complement experimental screening and selection techniques by predicting libraries of low-energy sequences compatible with a desired structure and function. Incorporating backbone flexibility in computational design allows conformational adjustments that should broaden the range of predicted low-energy sequences. Here, we evaluate computational predictions of sequence libraries from different protocols for modeling backbone flexibility using the complex between the therapeutic antibody Herceptin and its target human epidermal growth factor receptor 2 (HER2) as a model system. Within the program RosettaDesign, three methods are compared: The first two use ensembles of structures generated by Monte Carlo protocols for near-native conformational sampling: kinematic closure (KIC) and backrub, and the third method uses snapshots from molecular dynamics (MD) simulations. KIC or backrub methods were better able to identify the amino acid residues experimentally observed by phage display in the Herceptin-HER2 interface than MD snapshots, which generated much larger conformational and sequence diversity. KIC and backrub, as well as fixed backbone simulations, captured the key mutation Asp98Trp in Herceptin, which leads to a further threefold affinity improvement of the already subnanomolar parental Herceptin-HER2 interface. Modeling subtle backbone conformational changes may assist in the design of sequence libraries for improving the affinity of antibody-antigen interfaces and could be suitable for other protein complexes for which structural information is available.