Temporal and Spatial Requirement of EMF1 Activity for Arabidopsis Vegetative and Reproductive Development

EMBRYONIC FLOWER （EMF） genes are required to maintain vegetative development via repression of flower homeotic genes in Arabidopsis. Removal of EMF gene function caused plants to flower upon germination, producing abnormal and sterile flowers. The pleiotropic effect of ernfl mutation suggests its requirement for gene programs involved in diverse developmental processes. Transgenic plants harboring EMF1 promoter：：glucuronidase （GUS） reporter gene were generated to investigate the temporal and spatial expression pattern of EMF1. These plants displayed differential GUS activity in vegetative and flower tissues, consistent with the role of EMF1 in regulating multiple gene programs. EMFI：：GUS expression pattern in emf mutants suggests organ-specific auto-regulation. Sense- and antisense （as） EMF1 cDNA were expressed under the control of stage- and tissue-specific promoters in transgenic plants. Characterization of these transgenic plants showed that EMF1 activity is required in meristematic as well as differentiating tissues to rescue emf mutant phenotype. Temporal removal or reduction of EMF1 activity in the embryo or shoot apex of wild-type seedlings was sufficient to cause early flowering and terminal flower formation in adult plants. Such reproductive cell memory is reflected in the flower MADS-box gene activity expressed prior to flowering in these early flowering plants. However, temporal removal of EMF1 activity in flower meristem did not affect flower development. Our results are consistent with EMF1＇s primary role in repressing flowering in order to allow for vegetative growth.     

Engineered bromodomains to explore the acetylproteome

MS‐based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti‐acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine‐containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot‐to‐lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.     

Quality Control Test for Sequence-Phenotype Assignments

Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10–20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at http://bis.ifc.unam.mx/en/software/chispas.     

The Homeodomain Iroquois Proteins Control Cell Cycle Progression and Regulate the Size of Developmental Fields

During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins play a key, evolutionarily conserved, role in territorial specification. Here we show that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. We demonstrate that physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, our results provide a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors.     

Application of oncoproteomics to aberrant signalling networks in changing the treatment paradigm in acute lymphoblastic leukaemia

Oncoproteomics is an important innovation in the early diagnosis, management and development of personalized treatment of acute lymphoblastic leukaemia (ALL). As inherent factors are not completely known – e.g. age or family history, radiation exposure, benzene chemical exposure, certain viral exposures such as infection with the human T‐cell lymphoma/leukaemia virus‐1, as well as some inherited syndromes may raise the risk of ALL – each ALL patient may modify the susceptibility of therapy. Indeed, we consider these unknown inherent factors could be explained via coupling cytogenetics plus proteomics, especially when proteins are the ones which play function within cells. Innovative proteomics to ALL therapy may help to understand the mechanism of drug resistance and toxicities, which in turn will provide some leads to improve ALL management. Most important of these are shotgun proteomic strategies to unravel ALL aberrant signalling networks. Some shotgun proteomic innovations and bioinformatic tools for ALL therapies will be discussed. As network proteins are distinctive characteristics for ALL patients, unrevealed by cytogenetics, those network proteins are currently an important source of novel therapeutic targets that emerge from shotgun proteomics. Indeed, ALL evolution can be studied for each individual patient via oncoproteomics.     

Dietary Total Antioxidant Capacity and Colorectal Cancer in the Italian EPIC Cohort

Background

Colorectal cancer is the third most common cancer worldwide. Diet has been hypothesized as involved in colorectal cancer etiology, but few studies on the influence of total dietary antioxidant intake on colorectal cancer risk have been performed.

Methods

We investigated the association between colorectal cancer risk and the total antioxidant capacity (TAC) of the diet, and also of intake of selected antioxidants, in 45,194 persons enrolled in 5 centers (Florence, Naples, Ragusa, Turin and Varese) of the European Prospective Investigation into Cancer and Nutrition (EPIC) Italy study. TAC was estimated by the Trolox equivalent antioxidant capacity (TEAC) assay. Hazard ratios (HRs) for developing colorectal cancer, and colon and rectal cancers separately, adjusted for confounders, were estimated for tertiles of TAC by Cox modeling, stratifying by center.

Results

Four hundred thirty-six colorectal cancers were diagnosed over a mean follow-up of 11.28 years. No significant association between dietary TAC and colorectal cancer incidence was found. However for the highest category of TAC compared to the lowest, risk of developing colon cancer was lower (HR: 0.63; 95% CI: 0.44–0.89, P trend: 0.008). By contrast, increasing TAC intake was associated with significantly increasing risks of rectal cancer (2nd tertile HR: 2.09; 95%CI: 1.19–3.66; 3rd tertile 2.48 95%CI: 1.32–4.66; P trend 0.007). Intakes of vitamin C, vitamin E, and ß-carotene were not significantly associated with colorectal cancer risk.

Conclusions

Further prospective studies are needed to confirm the contrasting effects of high total antioxidant intake on risk of colon and rectal cancers.     

GSE4, a Small Dyskerin- and GSE24.2-Related Peptide,Induces Telomerase Activity,Cell Proliferation and Reduces DNA Damage,Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells

Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.     

The p13 protein of human T cell leukemia virus type 1 (HTLV-1) modulates mitochondrial membrane potential and calcium uptake

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K⁺) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Δψ) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's α-helical domain that were previously shown to be essential for its activity in in vitro assays. As Δψ is known to control mitochondrial calcium (Ca²⁺) uptake, we next analyzed the effect of p13 on Ca²⁺ homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca²⁺ uptake. These observations suggest that p13 might control key processes regulated through Ca²⁺ signaling such as activation and death of T cells, the major targets of HTLV-1 infection.