Maria Rosario Fernandez-Fernandez

Biotechnological aspects of plum pox virus.

Plum pox potyvirus (PPV), the causal agent of a devastating disease that affects stone fruit trees, is becoming a target of intense studies intended both to fight against viral infection and to develop practical applications based on the current knowledge of potyvirus molecular biology. This review focuses on biotechnological aspects related to PPV, such as novel diagnostic techniques that facilitate detection and typing of virus isolates, strategies to implement pathogen-derived resistance through plant transformation, the potential use of genetic elements derived from the virus, and the recent development of PPV-based expression vectors.

Impaired ATF6α processing, decreased Rheb and neuronal cell cycle re-entry in Huntington's disease.

The endoplasmic reticulum-stress response is induced in several neurodegenerative diseases and in cellular models of Huntingtons disease. However, here we report that the processing of ATF6α to its active nuclear form, one of the three branches of endoplasmic reticulum-stress activation, is impaired in both animal models and Huntingtons disease patients. ATF6α has been reported to be essential for the survival of dormant tumour cells that, like neurons, are arrested in the G0-G1 phase of the cell cycle. This effect is mediated by the small GTPase Rheb (Ras-homologue enriched in brain). Our results suggest that the ATF6α/Rheb pathway is altered in Huntingtons disease as the decrease in ATF6α processing is accompanied by a decrease in the accumulation of Rheb. These alterations correlate with the aberrant accumulation of cell cycle re-entry markers in post-mitotic neurons which is accompanied by death of a subset of neurons.

Hsp70: a master regulator in protein degradation

Proteostasis, the controlled balance of protein synthesis, folding, assembly, trafficking and degradation, is a paramount necessity for cell homeostasis. Impaired proteostasis is a hallmark of ageing and of many human diseases. Molecular chaperones are essential for proteostasis in eukaryotic cells, and their function has traditionally been linked to protein folding, assembly and disaggregation. More recent findings suggest that chaperones also contribute to key steps in protein degradation. In particular, Hsp70 has an essential role in substrate degradation through the ubiquitin–proteasome system, as well as through different autophagy pathways. Accumulated knowledge suggests that the fate of an Hsp70 substrate is dictated by the combination of partners (cochaperones and other chaperones) that interact with Hsp70 in a given cell context.

The relevance of protein–protein interactions for p53 function: the CPE contribution

The relevance of p53 as a tumour suppressor is evident from the fact that more than 50% of the human cancers hold mutations in the gene coding for p53, and of the remaining cancers a considerable number have alterations in the p53 pathway. From its discovery 30 years ago, the importance of p53 as an essential transcription factor for tumour suppression has become clear. More recently, new and seemingly diverse roles of p53 have been discovered. It soon became clear that protein-protein interactions play an important role in the regulation of the p53 function at different levels. Here we review the contribution by Prof. Fersht and his group towards understanding the basis and functional relevance of p53 protein-protein interactions, and the important role that protein science, biophysics and structural biology have played in the science produced in the Centre for Protein Engineering over the years.

3D electron tomography of brain tissue unveils distinct Golgi structures that sequester cytoplasmic contents in neurons

ABSTRACT Macroautophagy is morphologically characterized by autophagosome formation. Autophagosomes are double-membraned vesicles that sequester cytoplasmic components for further degradation in the lysosome. Basal autophagy is paramount for intracellular quality control in post-mitotic cells but, surprisingly, the number of autophagosomes in post-mitotic neurons is very low, suggesting that alternative degradative structures could exist in neurons. To explore this possibility, we have examined neuronal subcellular architecture by performing three-dimensional (3D) electron tomography analysis of mouse brain tissue that had been preserved through high-pressure freezing. Here, we report that sequestration of neuronal cytoplasmic contents occurs at the Golgi complex in distinct and dynamic structures that coexist with autophagosomes in the brain. These structures are composed of several concentric double-membraned layers that appear to be formed simultaneously by the direct bending and sealing of discrete Golgi stacks. These structures are labelled for proteolytic enzymes, and lysosomes and late endosomes are found in contact with them, leading to the possibility that the sequestered material could be degraded inside them. Our findings highlight the key role that 3D electron tomography, together with tissue rapid-freezing techniques, will have in gaining new knowledge about subcellular architecture. Summary: The power of new 3D imaging technologies, and optimal preservation of tissue samples, has helped to identify previously uncharacterized Golgi structures that sequester cytoplasmic material in neurons.

Proteinases Involved in Plant Virus Genome Expression

Publisher Summary This chapter discusses the proteinases involved in plant virus genome expression. The chapter focuses on virus-encoded proteinases. It gives an overall view of the use of proteolytic processing by different plant virus groups for the expression of their genomes. It also discusses that the development of full-length cDNA clones from which infectious transcripts can be produced either in vitro or in vivo, has facilitated the functional analysis of the plant virus proteinases. In spite of the high specificity of the viral proteinases, cellular substrates for animal virus proteinases have been described in this chapter. The activity of the viral proteinases can interfere with important cellular processes to favor virus replication. The recent use of proteinase inhibitors in AIDS therapy has emphasized the convenience of virus-encoded proteinases as targets of antiviral action. A mutant protein able to inhibit the activity of the TEV proteinase by manipulation of the α2-macroglobulin bait region was designed by Van Rompaey.

Hsp70 chaperone: a master player in protein homeostasis

Protein homeostasis (proteostasis) is an essential pillar for correct cellular function. Impairments in proteostasis are encountered both in aging and in several human disease conditions. Molecular chaperones are important players for proteostasis; in particular, heat shock protein 70 (Hsp70) has an essential role in protein folding, disaggregation, and degradation. We have recently proposed a model for Hsp70 functioning as a “multiple socket”. In the model, Hsp70 provides a physical platform for the binding of client proteins, other chaperones, and cochaperones. The final fate of the client protein is dictated by the set of Hsp70 interactions that occur in a given cellular context. Obtaining structural information of the different Hsp70-based protein complexes will provide valuable knowledge to understand the functional mechanisms behind the master role of Hsp70 in proteostasis. We additionally evaluate some of the challenges for attaining high-resolution structures of such complexes.