The Proto-Oncogene Int6 Is Essential for Neddylation of Cul1 and Cul3 in Drosophila

Int6 is a proto-oncogene implicated in various types of cancer, but the mechanisms underlying its activity are not clear. Int6 encodes a subunit of the eukaryotic translation initiation factor 3, and interacts with two related complexes, the proteasome, whose activity is regulated by Int6 in S. pombe, and the COP9 signalosome. The COP9 signalosome regulates the activity of Cullin-Ring Ubiquitin Ligases via deneddylation of their cullin subunit. We report here the generation and analysis of two Drosophila mutants in Int6. The mutants are lethal demonstrating that Int6 is an essential gene. The mutant larvae accumulate high levels of non-neddylated Cul1, suggesting that Int6 is a positive regulator of cullin neddylation. Overexpression in Int6 in cell culture leads to accumulation of neddylated cullins, further supporting a positive role for Int6 in regulating neddylation. Thus Int6 and the COP9 signalosome play opposing roles in regulation of cullin neddylation

Aspergillus Bibliography 2000

This bibliography attempts to cover genetical and biochemical publications on Aspergillus nidulans and also includes selected references to related species and topics

Protein ubiquitination in auxin signaling and transport

What makes plant shoots grow towards the light, and plant roots grow down into the soil? This was a question that Charles Darwin asked himself, and his experiments more than a century ago to find the answer laid the basis for the identification of the growth hormone auxin. Auxin, or indole-3-acetic acid (IAA), directs plant growth and development through its polar cell-to-cell transport-driven asymmetric distribution. Cellular IAA concentrations determine cell division, -elongation and -differentiation by facilitating the degradation of the Aux/IAA repressor proteins and thus inducing gene expression. The presumed pathway for the programmed degradation of proteins involves the attachment of a protein called ubiquitin, leading to recognition and destruction by a molecular complex called the proteasome. Here we investigated the role of protein ubiquitination and degradation in auxin action. First, we provided evidence for the longstanding paradigm that Aux/IAA proteins are ubiquitinated prior to their proteasomal degradation. At the same time we showed that ubiquitin labeling is not necessarily required for proteasomal degradation of plant proteins. Moreover, we showed that a regulator of auxin transport polarity is also involved in fine tuning auxin responses through the ubiquitin pathway. Our results place protein ubiquitination at a central position in auxin biology and thus in the movement of plants.UBL - phd migration 201

Identification of novel regulators of COP1-controlled morphogenesis in Arabidopsis thaliana

In Arabidopsis thaliana, COP1 is an essential element of light signal transduction acting downstream of photoreceptors and upstream of light-regulated gene expression. The COP1 protein acts as part of an E3 ligase complex to suppress photomorphogenic gene expression by ubiquitin-dependent degradation of light-regulated transcription factors. In dark-grown seedlings, the repression of photomorphogenesis involves the inhibition of hypocotyl growth, anthocyanin accumulation, expression of light-responsive genes, differentiation of etioplasts and prevention of apical hook formation. Loss of COP1 function leads to a pleiotropic phenotype comprising of constitutive photomorphogenesis in the dark and resulting in a post-germination growth arrest. The vegetative growth arrest of cop1 mutants, a possible role of COP1 concerning the cell cycle and the molecular factors regulating the nucleocytoplasmic partitioning of COP1 exemplify aspects of COP1 function and regulation that are poorly understood until now. This work aimed at the identification of regulators of COP1-controlled morphogenesis to contribute to a better dissection of the latter. In yeast two hybrid screenings (YTH) 32 new interaction candidates for COP1 were identified and a purpose oriented selection was performed. In order to select a putative regulator of COP1, all COP1 and additional DET1 interaction candidates were integrated in a network of published interactors. Out of the network and the screening results PAP2 (PRODUCTION OF ANTHOCYAN PIGMENT) was identified and selected as a putative new target. MID (= MIDGET) was selected as a putative new regulator of COP1, respectively. MID is a part of the topoisomerasis VI (TOPOVI) complex that is needed to complete more than two endocycles in plant cells. This work provides evidence for a physical interaction of MID and PAP2 with COP1. In addition, a new YTH-based domain mapping method was developed and used to identify so far unknown domains of PAP2 for the interaction with COP1 and for COP1 for the interaction with MID and TOPOVI components. Similar to cop1, mid and topoisomerase VI mutants exhibited all aspects of constitutive pohotomorphogenesis in the dark. Double mutant analysis indicated that MID is not a target of COP1. In infiltrated leaves of Nicotiana benthamina, the presence of MID is needed for COP1 to form a high number of subnuclear foci. MID and the TOPOVI were shown to be essential regulators of COP1 function probably by stabilising COP1 and thereby adding a new cell-cycle related factor to the regulation of COP1 activity. The functional relevance of the MID-COP1 interaction was proven by analysing phenotypes of the single mutants and genetic interaction. First evidence positioning MID in a SPA1 and phyA-dependent complex or pathway were obtained by the verification of the SPA1-MID interaction via BiFC, co-purification of MID with phyA and analysis of the protein stability of MID depending on light quality. Finally it was found that mid and topoVI mutants phenocopy det1-1 mutants and overexpressor lines of the C-termini of CRY1 and CRY2, possibly providing a new link to crosstalk between red and blue light mediated signaling

Caracterización molecular y funcional del gen PATHOGEN AND CIRCADIAN CONTROLLED 1 (PCC1) en Arabidopsis thaliana

Las plantas son capaces de modificar los patrones de desarrollo tras percibir ciertos tipos de estrés. En Arabidopsis, se identificó PCC1 como un regulador positivo de la transición floral en respuesta al estrés generado por irradiación con luz UV-C. El análisis de plantas transgénicas pPCC1::GUS muestra que PCC1 se expresa durante los primeros días de desarrollo en estomas y haces vasculares de cotiledones. En hojas verdaderas en formación se detecta tinción GUS en su parte basal, incluyendo los haces vasculares, y se va extendiendo completamente a toda la superficie de hojas completamente formadas. Líneas que expresan construcciones de RNAi para PCC1 (iPCC1) presentan reducidos niveles de FT y, consecuentemente, una floración más tardía. El mecanismo por el cual PCC1 podría regular la transición floral parece estar relacionado con alteraciones en la transmisión de la señal por luz. Concomitantemente, las plantas iPCC1 muestran fenotipos parcialmente escotomorfogénicos en los distintos tipos de luz ensayados de forma independiente de la acumulación y señalización de GAs. El transcriptoma diferencial de plantas iPCC1 versus plantas silvestres muestra una clara implicación de PCC1 en procesos relacionados con defensa. De acuerdo con este hecho, hemos observado que las plantas iPCC1 son más susceptibles a la infección con el oomiceto hemi-biotrofo Phytophtora brassicae y más resistentes al hongo necrotrofo Botrytis cinerea. Además, las líneas iPCC1 presentan una regulación al alza de genes de respuesta a ABA, y una mayor sensibilidad a esta fitohormona para los distintos fenotipos analizados. Finalmente, entre los genes alterados en las líneas iPCC1 se observa una sobrerepresentación de genes implicados en el metabolismo y en el transporte de lípidos. La pérdida de función de PCC1 conlleva una reducción del 70% en los niveles de fosfatidilinositol, y en menor medida de otros tipos de lípidos polares como la fosfatidilserina o la fosfatidilcolina. Además, el análisis de la composición de ácidos grasos de cada tipo de lípidos polares revela un mayor grado de insaturación de sus cadenas laterales, fundamentalmente en la fosfatidilserina y el fosfatidilinositol. PCC1 es una proteína asociada a la membrana plasmática por su extremo carboxiterminal, el cual es responsable además de la formación de homodímeros. Aunque queda por dilucidiar los mecanismos por los cuales PCC1 puede regular procesos tan dispares molecularmente como la respuesta a patógenos y la transición floral, hemos observado que PCC1 interacciona con la subunidad CSN5 del signalosoma (CSN), lo que sugiere que PCC1 podría actuar como un regulador de la función de CSN, y en última instancia, de la degradación de proteínas por ubiquitinación.Mir Moreno, R. (2013). Caracterización molecular y funcional del gen PATHOGEN AND CIRCADIAN CONTROLLED 1 (PCC1) en Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/29751TESI

Role and Regulation of Autophagy During Developmental Cell Death in Drosophila Melanogaster

Two prominent morphological forms of programmed cell death occur during development, apoptosis and autophagic cell death. Improper regulation of cell death can lead to a variety of diseases, including cancer. Autophagy is required for survival in response to starvation, but has also been associated with cell death. It is unclear how autophagy is regulated under specific cell contexts in multi-cellular organisms, and what may distinguish autophagy function during cell survival versus cell death. Autophagic cell death is characterized by cells that die in synchrony, with autophagic vacuoles in the cytoplasm, and phagocytosis of the dying cells is not observed. However, little is known about this form of cell death. Autophagic cell death is observed during mammalian development, during regression of the corpus luteum and involution of the mammary and prostate glands. Autophagic cell death is also observed during development of the fruitfly Drosophila melanogaster, during larval salivary gland cell death. Drosophila is an excellent genetic model system to study developmental cell death in vivo. Cells use two main catabolic processes to degrade and recycle cellular contents, the ubiquitin/proteasome system (UPS) and autophagy. Here I investigate the role of the UPS and autophagy in developmental cell death using Drosophila larval salivary glands as an in vivo model. Proteasome inhibitors are being used in anti-cancer therapies; however the cellular effects of proteasome inhibition have not been studied in vivo. Here I demonstrate that the UPS is impaired during developmental cell death in vivo. Taking a proteomics approach to identify proteins enriched in salivary glands during developmental cell death and in response to proteasome impairment, I identify several novel genes required for salivary gland cell death, including Cop9 signalsome subunit 6 and the engulfment receptor Draper. Here I show that the engulfment receptor Draper is required for salivary gland degradation. This is the first example of an engulfment factor that is autonomously required for self-clearance. Surprisingly, I find that Draper is cell-autonomously required for autophagy during cell death, but not for starvation-induced autophagy. Draper is the first factor to be identified that genetically distinguishes autophagy that is associated with cell death from cell survival