RESUMO
Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Oogênese/genética , Ribonucleoproteínas/genética , Ubiquitina-Proteína Ligases/genética , Animais , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Homeostase/genética , Longevidade/genética , Neurônios/citologia , Neurônios/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Transição de Fase , Profilinas/genética , Profilinas/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. Here we show that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. We demonstrate that this PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1α (HP1α). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies.
Assuntos
Proteínas de Drosophila/metabolismo , Heterocromatina/metabolismo , Lamina Tipo B/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase/metabolismo , Estresse Mecânico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Células Cultivadas , Homólogo 5 da Proteína Cromobox/genética , Homólogo 5 da Proteína Cromobox/metabolismo , Elementos de DNA Transponíveis/genética , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Humanos , Lamina Tipo B/química , Camundongos , Camundongos Endogâmicos C57BL , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/genética , Neocórtex/citologia , Neocórtex/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Membrana Nuclear/química , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
In this chapter, we describe techniques to detect ubiquitination events occurring in vivo. We focus on methodologies capable of preserving and detecting target protein ubiquitination in physiological conditions, without overexpressing a tagged version of ubiquitin. We provide detailed protocols for mammalian and Drosophila melanogaster systems using linkage-specific antibodies against ubiquitin. We analyze immunoblotting and immunofluorescence approaches as well as an ELISA-based quantitative approach, which allows comparing more samples and conditions. Finally, we describe the use of in vitro ubiquitination assays with purified proteins that allows a direct analysis of the molecular mechanisms behind specific E3 activity.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Ubiquitinação , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células HeLa , Humanos , Immunoblotting/métodos , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Ubiquitination plays a pivotal role in most cellular processes and is critical for protein degradation and signalling. E3 ligases are the matchmakers in the ubiquitination cascade, responsible for substrate recognition and modification with specific polyubiquitin chains. Until recently, it was not clear how the catalytic activity of E3s is modulated, but major recent studies on HECT E3 ligases is filling this void. These enzymes appear to be held in a closed, inactive conformation, which is relieved by biochemical manoeuvres unique to each member, thus ensuring exquisite regulation and specificity of the enzymes. The new advances and their significance to the function of HECT E3s are described here, with a particular focus on the Nedd4 family members.