The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation

Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Deciphering the biological functions of F-box proteins through the use of Parallel Adaptor Capture (PAC) proteomics.

The timely and selective proteasomal degradation of proteins is important for the maintenance of proper cellular processes. Prior to proteasomal degradation, proteins destined for degradation are polyubiquitiylated by ubiquitin ligases (E3s). The SKP1-CUL1-F-box protein (SCF) complex is a member of the cullin-RING ligase (CRL) superfamily of modular, multi-protein E3s, in which the F-box protein (FBP) acts as a substrate specificity factor for the recruitment of substrates to the SCF complex. Many of the 69 human FBPs remain uncharacterized. From our current knowledge of FBPs, we know that they regulate a myriad and diverse set of cellular processes and their misregulation is associated with diseases, including cancer. Though many approaches exist for the identification of SCF substrates and/or interactors, most existing genetic and quantitative proteomic methods are not capable of adaptor identification, while interaction proteomic approaches are generally performed in a low throughput manner. To facilitate our characterization of FBPs, we have developed a novel, facile proteomic approach for the identification of interactors, including substrates, of FBPs. In this method, FBPs, which together with SKP1 form the adaptor subunit of the SCF complex, are individually expressed in cells. These cells are subsequently exposed to 3 conditions: left untreated, treated with MLN4924 (neddylation inhibitor) or treated with Bortezomib (proteasome inhibitor). After treatment, cells are lysed, and the lysates are affinity-purified and processed in parallel for proteomic analysis. This approach, which we have named Parallel Adaptor Capture (PAC) proteomics, was successfully applied for the characterization of three novel FBP/substrate interactions: FBXW11 (β-TrCP2)/DEPTOR (Appendix 1), FBXL17/BACH1 (Chapter 2) and FBXO22/KDM4A (Chapter 3). Besides the characterization of these FBPs, PAC proteomics was performed on 19 leucine-rich repeats containing FBPs, the FBXLs, identifying 230 high confidence interacting proteins, including known regulatory proteins and substrates. Deciphering the biological context and significance of these interactions will allow us to understand the importance of FBXLs in the cellular processes in which they regulate. Since CRLs require neddylation to efficiently ubiquitylate their substrates and most CRL substrates are degraded by the proteasome, PAC proteomics, in principle, can also be applied to other CRL adaptors

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field