MEKK5 Interacts with and Negatively Regulates the E3 Ubiquitin Ligase NEDD4 for Mediating Lung Cancer Cell Migration

Our previous studies have shown that the HECT E3 ubiquitin ligase NEDD4 and kinase MEKK5 both play an essential role in lung cancer migration. A report predicts that MEKK5 may be ubiquitinated by NEDD4; however, interaction of MEKK5 with NEDD4 and ubiquitination of MEKK5 by NEDD4 have not been characterized. In this report, we show that NEDD4 interacts with MEKK5 through a conserved WW3 domain by the co-immunoprecipitation and the GST-pulldown assays. The ubiquitination assay indicates that MEKK5 is not a ubiquitination substrate of NEDD4, but negatively regulates NEDD4-mediated ubiquitination. Furthermore, overexpression of MEKK5 significantly reduced the NEDD4-promoted lung cancer cell migration. Taken together, our studies have defined an inhibitory role of MEKK5 in regulation of NEDD4-mediated ubiquitination.

miR-101-3p Contributes to α-Synuclein Aggregation in Neural Cells through the miR-101-3p/SKP1/PLK2 Pathway

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive neuronal loss in different brain regions, including the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc). The aggregation of α-synuclein (α-Syn) plays an essential role in the progression of PD-related neuron toxicity. In this study, bioinformatic analysis was used to confirm differentially expressed genes between patients with PD and healthy donors. Immunofluorescence was used to study the aggregation of α-Syn. Flow cytometry was used to confirm the apoptosis of neurons. Western blot was used to investigate the underlying mechanism. Coimmunoprecipitation (co-IP) was used to verify the interaction between proteins. Luciferase activity assay was used to confirm the target gene of miRNA. In vitro protein ubiquitination assay was used to ascertain the role of S-phase kinase-associated protein 1 (SKP1) on the ubiquitination processes of polo-like kinase 2 (PLK2). The result indicated that miR-101-3p was overexpressed in the substantia nigra of the postmortem brains of patients with PD. The underlying role was investigated in the SH-SY5Y cell line. The overexpression of α-Syn did not result in toxicity or aggregation. However, the co-overexpression of miR-101-3p and α-Syn promoted aggregation and neuron toxicity. Luciferase activity assay indicated that SKP1 is a target gene of miR-101-3p. The co-IP experiment confirmed that SKP1 could directly interact with PLK2. In vitro protein ubiquitination assay confirmed that SKP1 could promote the ubiquitination and subsequent protein degradation of PLK2. We also observed that the cotransfection of short hairpin RNA that targets PLK2 and α-Syn overexpression plasmid results in the endoplasmic reticulum stress of neurons. Our results collectively provide evidence that miR-101-3p contributes to α-Syn aggregation in neurons through the miR-101-3p/SKP1/PLK2 pathway.

Silencing of TRIM11 suppresses the tumorigenicity of chordoma cells through improving the activity of PHLPP1/AKT

Background Tripartite motif-containing protein 11 (TRIM11), a member of RING family of E3 ubiquitin ligases, is identified as an oncogene in certain human tumors. However, the detailed biological function of TRIM11 in chordoma is still unclear. The purpose of present research is to explore the role of TRIM11 in human chordoma cells. Methods TRIM11 was induced silencing and overexpression in human chordoma cells using RNA interference (RNAi) and lentiviral vector. qRT-PCR and western blot were used to determine gene expression in chordomas cells. Meanwhile, cell counting kit-8 (CCK-8) assay was used to examine the cell proliferation rate. Flow cytometry analysis was performed to quantify the cell apoptosis rate. Results We identified that TRIM11 was upregulated in chordomas tissues. Moreover, TRIM11 presented pro-proliferation and anti-apoptosis function in chordoma cells. Further, LY294002, a specific AKT inhibitor, was utilized to examine the connection between TRIM11 and AKT in human chordoma cells. Importantly, our findings elucidated that TRIM11 promoted the growth of chordoma cells and involved in AKT signaling. Much more importantly, knockdown of TRIM11 significantly upregulated the translation of PH domain leucine-rich repeats protein phosphatase 1 (PHLPP1), whereas did not affect its transcription. Results that obtained from co-immunoprecipitation (Co-IP) and ubiquitination assay demonstrated TRIM11 interacted with PHLPP1 and promoted its ubiquitination in chordoma cells. Moreover, overexpression of PHLPP1 inhibited the phosphorylation of AKT in human chordomas cells. These results suggested that TRIM11 mediated the post-translation modification of PHLPP1 and was a novel component in PHLPP1/AKT signaling pathway in human chordoma cells. Conclusions Taken together, the present research not only enhanced the understanding of TRIM11 but also indicated its potential target and signaling pathway in human chordoma cells. Trial registration retrospectively registered.

A general in vitro assay to study enzymatic activities of the ubiquitin system

The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2 and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies on ubiquitination activities have been challenging. We here report a novel FRET-based assay to study the in vitro kinetics of ubiquitination. FRET is established between binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model E1, E2 and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates, and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus likely compatible with any E1-E2-E3 combinations.

Kir5.1 regulates Nedd4-2-mediated ubiquitination of Kir4.1 in distal nephron

Kir4.1/5.1 heterotetramer participates in generating the negative cell membrane potential in distal convoluted tubule (DCT) and plays a critical role in determining the activity of Na-Cl cotransporter (NCC). Kir5.1 contains a phosphothreonine motif at its COOH terminus (AA249–252). Coimmunoprecipitation showed that Nedd4-2 was associated with Kir5.1 in HEK293 cells cotransfected with Kir5.1 or Kir4.1/Kir5.1. GST pull-down further confirmed the association between Nedd4-2 and Kir5.1. Ubiquitination assay showed that Nedd4-2 increased the ubiquitination of Kir4.1/Kir5.1 heterotetramer in the cells cotransfected with Kir4.1/Kir5.1, but it has no effect on Kir4.1 or Kir5.1 alone. Patch-clamp and Western blot also demonstrated that coexpression of Nedd4-2 but not Nedd4-1 decreased K currents and Kir4.1 expression in the cells cotransfected with Kir4.1 and Kir5.1. In contrast, Nedd4-2 fails to inhibit Kir4.1 in the absence of Kir5.1 or in the cells transfected with the inactivated form of Nedd4-2 (Nedd4-2C821A). Moreover, the mutation of TPVT motif in the COOH terminus of Kir5.1 largely abolished the association of Nedd4-2 with Kir5.1 and abolished the inhibitory effect of Nedd4-2 on K currents in HEK293 cells transfected with Kir4.1 and Kir5.1 mutant (Kir5.1T249A). Finally, the basolateral K conductance in the DCT and Kir4.1 expression is significantly increased in the kidney-specific Nedd4-2 knockout or in Kir5.1 knockout mice in comparison to their corresponding wild-type littermates. We conclude that Nedd4-2 binds to Kir5.1 at the phosphothreonine motif of the COOH terminus, and the association of Nedd4-2 with Kir5.1 facilitates the ubiquitination of Kir4.1, thereby regulating its plasma expression in the DCT.

PARAQUAT TOLERANCE3 is an E3 ligase and acts as a negative regulator of oxidative stress response

Oxidative damage could be caused in plant cells when biotic and abiotic stresses are imposed. While the response to oxidative stress is well studied, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3) as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b may regulate the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. Moreover, PQT3 is able to recognize PRMT4b for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress.