RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARa receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies

Cop1 constitutively regulates c-Jun protein stability and functions as a tumor suppressor in mice

Biochemical studies have suggested conflicting roles for the E3 ubiquitin ligase constitutive photomorphogenesis protein 1 (Cop 1; also known as Rfwd2) in tumorigenesis, providing evidence for both the oncoprotein c-Jun and the tumor suppressor p53 as its targets. Here we present what we believe to be the first in vivo investigation of the role of Cop1 in cancer etiology. Using an innovative genetic approach to generate an allelic series of Cop1, we found that Cop1 hypomorphic mice spontaneously developed malignancy at a high frequency in the first year of life and were highly susceptible to radiation-induced lymphomagenesis. Further analysis revealed that c-Jun was a key physiological target for Cop1 and that Cop1 constitutively kept c-Jun at low levels in vivo and thereby modulated c-Jun/AP-1 transcriptional activity. Importantly, Cop1 deficiency stimulated cell proliferation in a c-Jun-dependent manner. Focal deletions of COP1 were observed at significant frequency across several cancer types, and COP1 loss was determined to be one of the mechanisms leading to c-Jun upregulation in human cancer. We therefore conclude that Cop1 is a tumor suppressor that functions, at least in part, by antagonizing c-Jun oncogenic activity. In the absence of evidence for a genetic interaction between Cop1 and p53, our data strongly argue against the use of Cop1-inhibitory drugs for cancer therapy

ArrayMAPPIT: a screening platform for human protein interactome analysis

Mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid technology to identify and characterize interactions of proteins with other proteins or organic molecules in living mammalian (human) cells. The method relies on complementation of a modified cytokine receptor complex. Protein interaction restores the signalling competence of the complex, which is monitored through the activation of a reporter gene. Here, we describe a protocol that has been recently developed to increase the utility of MAPPIT as a tool to identify novel interactions. In the ArrayMAPPIT assay, a collection of prey proteins which is arrayed in high-density microtiter plates is efficiently screened for interaction partners using reverse transfection into a bait-expressing cell pool

An incremental grey-box current regression model for anomaly detection of resistance mash seam welding in steel mills

Featured Application Our anomaly detection model will be used to monitor seam welding in the cold mill annealing line of the ArcelorMittal Ghent steel mill. Annealing and galvanization production lines in steel mills run continuously to maximize production throughput. As a part of this process, individual steel coils are joined end-to-end using mash seam welding. Weld breaks result in a production loss of multiple days, so non-destructive, data-driven techniques are used to detect and replace poor quality welds in real-time. Statistical models are commonly used to address this problem as they use data readily available from the welding machine and require no specialized equipment. While successful in finding anomalies, these statistical models do not provide insight into the underlying process and are slow to adapt to changes in the machine's or material's behavior. We combine knowledge-based and data-driven techniques to create an incremental grey-box welding current prediction model for detecting anomalous welds, resulting in a powerful and interpretable model. In this work, we detail our approach and show evaluation results on industrial welding data collected over a period of 15 months containing behavioral shifts attributed to machine maintenance. Due to its incremental nature, our model resulted in two-thirds fewer rejected welds compared to statistical models, thus greatly reducing production overhead. Grey-box modeling can be applied to other welding features or domains and results in models that are more desirable for the industry

The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells

RNA-protein interactions are essential for the regulation of mRNA and noncoding RNA functions and are implicated in many diseases, such as cancer and neurodegenerative disorders. A method that can detect RNA-protein interactions in living mammalian cells on a proteome-wide scale will be an important asset to identify and study these interactions. Here we show that a combination of the mammalian two-hybrid protein-protein detection method KISS (kinase substrate sensor) and the yeast RNA three-hybrid method, utilizing the specific interaction between the MS2 RNA and MS2 coat protein, is capable of detecting RNA-protein interactions in living mammalian cells. For conceptional proof we used the subgenomic flavivirus RNA (sfRNA) of the dengue virus (DENV), a highly structured noncoding RNA derived from the DENV genome known to target host cell proteins involved in innate immunity and antiviral defense, as bait. Using RNA-KISS, we could confirm the previously established interaction between the RNA-binding domain of DDX6 and the DENV sfRNA. Finally, we performed a human proteome-wide screen for DENV sfRNA-binding host factors, identifying several known flavivirus host factors such as DDX6 and PACT, further validating the RNA-KISS method as a robust and high-throughput cell-based RNA-protein interaction screening tool.status: publishe

VPS52 is a novel interaction partner of RNF41.

<p><b>(A)</b> Array MAPPIT screen result of the RNF41 C34S/H36Q bait against a library covering up to 8,500 preys shown as a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus <i>P</i> value. <b>(B)</b> MAPPIT analysis of HEK293T cells transiently cotransfected with a vector encoding an RNF41 or empty bait, a VPS52 prey plasmid and the pXP2d2-rPAP1-luciferase reporter plasmid, followed by 24 hours Epo stimulation or left untreated. The luciferase signal is expressed as fold induction (stimulated/non stimulated), relative to the signal of a control JAK2 binding prey ± s.d. of triplicate measurements. Western blotting verified Flag-tagged prey expression and β-actin levels (loading control). <b>(C)</b> AlphaScreen analysis of HEK293T cells transiently cotransfected with plasmids encoding E-tagged VPS52 and Flag-tagged RNF41 or SV40 large T antigen (SVT) as an irrelevant protein (mock). After 48 hours, cells were lysed and protein interactions were detected with the AlphaScreen FLAG^TM (M2) detection kit (PerkinElmer Life Sciences) by generation of a luminescent signal, displayed as absolute light units. Western blotting shows E and Flag-tagged protein expression and a β-actin loading control. <b>(D)</b> Co-immunoprecipitation of endogenous VPS52 and RNF41. HEK293T lysates were immunoprecipitated with anti-VPS52 or a control normal rabbit IgG. The immune complexes (first and second lane) and the input (third lane) were analyzed by immunoblotting with an antibody specific to RNF41 (middle panel). Anti-actin was used as a loading control <b>(E)</b> GST-pulldown of <i>Escherichia coli</i> BL21(DE3) cell lysates expressing GST-PGC1α (negative control, lane 2) or GST-RNF41 (lane 3) using glutathione sepharose beads and incubated with in vitro transcribed and translated Flag-tagged VPS52. Western blotting using anti-Flag and anti-GST antibodies reveals bead-bound protein complexes and expression of Flag-tagged VPS52 (lane 1). <b>(F)</b> Confocal microscopy of HeLa cells transfected with plasmids encoding either E-tagged VPS52 (left panel) or untagged RNF41 (middle panel), together with soluble IL5Rα plasmid as a control, or plasmids encoding E-tagged VPS52 and RNF41 (right panel). Cells were fixed and stained with DAPI (nuclei staining, blue), anti-Etag (secondary Alexa Fluor 488, green) and anti-RNF41 (secondary Alexa Fluor 568, red) antibodies. The inset shows a magnification of the boxed area. The white overlay represents the intersect between RNF41 and VPS52 with a threshold set on standard deviation using Volocity 6.3 software (Perkin Elmer, Waltham, MA, USA). Scale bar, 10μm. n = 3 or more for all performed experiments. <b>(G)</b> Co-immunoprecipitation analysis of HEK293T cells transiently cotransfected with plasmids encoding E-tagged RNF41 and Flag-tagged GARP/EARP subunits or soluble IL5Rα. Anti-Flag immunoprecipitates (right panel) and lysates (left panel) were visualized with anti-Flag, anti-Etag and anti-actin. <b>(H)</b> AlphaScreen analysis of HEK293T cells transiently cotransfected with plasmids encoding E-tagged RNF41 and Flag-tagged GARP/EARP subunits or SVT.</p