RESUMO
Protein phosphatase one (PP1) is a major eukaryotic serine/threonine protein phosphatase whose activity is controlled by targeting or regulatory subunits. Currently, very few plant protein phosphatase one regulatory subunits are known. Here, Arabidopsis GL2 EXPRESSION MODULATOR (GEM) was identified and confirmed as a protein phosphatase one binding partner. GEM is a phosphoprotein, contains a highly conserved phosphoinositide binding GRAM domain and a classic protein phosphatase one binding RVXF motif. Lipid overlays show GEM has the ability to interact with phosphoinositides through its GRAM domain. GEM is the first plant specific protein phosphatase one interactor to be discovered.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfatidilinositóis/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligação Proteica , Domínios Proteicos/genética , Proteína Fosfatase 1/metabolismoRESUMO
PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteína Fosfatase 1/química , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Filogenia , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Estruturas Vegetais/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/isolamento & purificação , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Toxic amyloid-ß (Aß) peptides aggregate into higher molecular weight assemblies and accumulate not only in the extracellular space, but also in the walls of blood vessels in the brain, increasing their permeability, and promoting immune cell migration and activation. Given the prominent role of the immune system, phagocytic blood cells may contact pathological brain materials. OBJECTIVE: To develop a novel method for early Alzheimer's disease (AD) detection, we used blood leukocytes, that could act as "sentinels" after trafficking through the brain microvasculature, to detect pathological amyloid by labelling with a conformationally-sensitive fluorescent amyloid probe and imaging with confocal spectral microscopy. METHODS: Formalin-fixed peripheral blood mononuclear cells (PBMCs) from cognitively healthy control (HC) subjects, mild cognitive impairment (MCI) and AD patients were stained with the fluorescent amyloid probe K114, and imaged. Results were validated against cerebrospinal fluid (CSF) biomarkers and clinical diagnosis. RESULTS: K114-labeled leukocytes exhibited distinctive fluorescent spectral signatures in MCI/AD subjects. Comparing subjects with single CSF biomarker-positive AD/MCI to negative controls, our technique yielded modest AUCs, which improved to the 0.90 range when only MCI subjects were included in order to measure performance in an early disease state. Combining CSF Aß42 and t-Tau metrics further improved the AUC to 0.93. CONCLUSION: Our method holds promise for sensitive detection of AD-related protein misfolding in circulating leukocytes, particularly in the early stages of disease.
Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/metabolismo , Diagnóstico Precoce , Corantes Fluorescentes/metabolismo , Leucócitos Mononucleares/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/sangue , Biomarcadores/líquido cefalorraquidiano , Encéfalo/patologia , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas tau/metabolismoRESUMO
The phosphoinositide-3-OH-kinase-related kinases (PIKKs) are atypical protein kinases exclusive to eukaryotes. They mediate the cellular response to a range of stresses, including genome and RNA surveillance and availability of nutrients for growth. Orthologues of five out of the six PIKK family members are present in plant genomes. Recent studies in plant PIKKs have revealed features unique to, and in common with, other PIKKs. This review summarizes the basic knowledge of these proteins in mammals and yeast in comparison with what is known for Arabidopsis and other plants.