Protein Microarrays for the Identification of Praja1 E3 Ubiquitin Ligase Substrates

Loc’h, Christian; Eddins, Michael J.; Strickler, J. Rudi

doi:10.1007/s12013-011-9180-x

Cited by 8 publications

(10 citation statements)

References 39 publications

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“…Furthermore, deletion of the region harbouring the PJA1 gene has been observed in patients with craniofrontonasal syndrome and may be associated with mild learning disabilities [12]. Several targets of Praja1-mediated polyubiquitination have already been identified, including the class II melanoma antigen (MAGE) family member NRAGE (neurotrophin receptor associated MAGE homologue), Smad3 and polycomb repressive complex 2 [13]–[15]. NRAGE (named Dlxin-1 in mouse and MAGE-D1 in human) may be of particular relevance for neuronal development; it is a multifunctional signalling molecule involved in – among others – neurotrophin (via p75 NTR ) and bone morphogenetic protein (BMP) signalling, as well as in UNC5H1 mediated cell adhesion, all of which are involved and appear to interact in neuronal differentiation [16]–[22].…”

Section: Introductionmentioning

confidence: 99%

The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

et al. 2013

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Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect.

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Section: Introductionmentioning

confidence: 99%

The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

et al. 2013

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“…Although refined recently by several modifications to increase efficiency in substrate identification (9,20,21), interaction-based methods are hindered by the transient, weak nature of the ubiquitin ligase-substrate interaction. To circumvent the limitations of interaction-based methods, high-throughput in vitro approaches, such as in vitro ubiquitination biochemistry coupled with protein microarrays, have proven to be successful at identifying ubiquitin ligase substrates (22,23). The use of in vitro -based methods is limited, however, to the content printed on protein arrays, limiting the substrate candidate pool.…”

Section: Introductionmentioning

confidence: 99%

Diggin′ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

Rubel

Schisler

Hamlett

et al. 2013

Cell Biochem Biophys

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The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing Tandem Ubiquitin Binding Entities (TUBE) technology, two-dimensional differential in gel electrophoresis (2-D DIGE), and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets.

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“…To date very little is known about the role of these proteins in the central nervous system. PRAJA-1 is a E2-dependent E3 ubiquitin ligase that is abundantly expressed in the brain (46), and has as its putative substrates the postsynaptic proteins Homer2, CAMK1G, EPHB3 and SEPT1 (47). PRAJA-1 has been implicated in learning and memory associated with fear conditioning (48).…”

Section: Discussionmentioning

confidence: 99%

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis

Stockton¹,

Gomes²,

Liu³

et al. 2015

Molecular & Cellular Proteomics

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Despite its efficacy, the use of morphine for the treatment of chronic pain remains limited because of the rapid development of tolerance, dependence and ultimately addiction. These undesired effects are thought to be because of alterations in synaptic transmission and neuroplasticity within the reward circuitry including the striatum. In this study we used subcellular fractionation and quantitative proteomics combined with computational approaches to investigate the morphine-induced protein profile changes at the striatal postsynaptic density. Over 2,600 proteins were identified by mass spectrometry analysis of subcellular fractions enriched in postsynaptic density associated proteins from saline or morphine-treated striata. Among these, the levels of 34 proteins were differentially altered in response to morphine. These include proteins involved in G-protein coupled receptor signaling, regulation of transcription and translation, chaperones, and protein degradation pathways. The altered expression levels of several of these proteins was validated by Western blotting analysis. Using Genes2Fans software suite we connected the differentially expressed proteins with proteins identified within the known background protein-protein interaction network. This led to the generation of a network consisting of 116 proteins with 40 significant intermediates. To validate this, we confirmed the presence of three proteins predicted to be significant intermediates: caspase-3, receptor-interacting serine/threonine protein kinase 3 and NEDD4 (an E3-ubiquitin ligase identified as a neural precursor cell expressed developmentally down-regulated protein 4). Because this morphine-regulated network predicted alterations in proteasomal degradation, we examined the global ubiquitination state of postsynaptic density proteins and found it to be substantially altered. Together, these findings suggest a role for protein degradation and for the ubiquitin/proteasomal system in the etiology of opiate dependence and addiction. Morphine and other opiates are the drugs of choice for the treatment of both severe and chronic pain. However, the utility of these compounds in the clinical setting is limited because of the rapid development of tolerance, physical dependence and addiction. The underlying cellular and molecular alterations through which chronic opiate exposure results in the persistent behavioral phenomenon of addiction remain poorly understood. However, there is evidence suggesting that the molecular composition of synapses within the reward circuitry of the central nervous system, particularly in the striatum, may be significantly altered (1). Moreover, given the critical involvement of the striatum in translating emotional or rewarding stimuli into motivated behaviors, alterations in this region induced by morphine and other abused drugs could contribute significantly to the pathophysiological responses at the heart of addiction (2). Although the debate surrounding the molecular and cellular mechanisms by which repeated drug adminis...

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Protein Microarrays for the Identification of Praja1 E3 Ubiquitin Ligase Substrates

Cited by 8 publications

References 39 publications

The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

Diggin′ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis

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