Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junctions have a wide diversity of physiological functions, playing critical roles in both excitable and non-excitable tissues. Gap junction channels are formed by integral membrane proteins called connexins. Inherited or acquired alterations in connexins are associated with numerous diseases, including heart failure, neuropathologies, deafness, skin disorders, cataracts and cancer. Gap junctions are highly dynamic structures and by modulating the turnover rate of connexins, cells can rapidly alter the number of gap junction channels at the plasma membrane in response to extracellular or intracellular cues. Increasing evidence suggests that ubiquitination has important roles in the regulation of endoplasmic reticulum-associated degradation of connexins as well as in the modulation of gap junction endocytosis and post-endocytic sorting of connexins to lysosomes. In recent years, researchers have also started to provide insights into the physiological roles of connexin ubiquitination in specific tissue types. This review provides an overview of the advances made in understanding the roles of connexin ubiquitination in the regulation of gap junction intercellular communication and discusses the emerging physiological and pathophysiological implications of these processes.

NBR1 was discovered as an autophagy receptor not long after the first described vertebrate autophagy receptor p62/SQSTM1. Since then, p62 has currently been mentioned in >10,000 papers on PubMed, while NBR1 is mentioned in <350 papers. Nonetheless, evolutionary analysis reveals that NBR1, and likely also selective autophagy, was present already in the last eukaryotic common ancestor (LECA), while p62 appears first in the early Metazoan lineage. Furthermore, yeast-selective autophagy receptors Atg19 and Atg34 represent NBR1 homologs. NBR1 is the main autophagy receptor in plants that do not contain p62, while most animal taxa contain both NBR1 and p62. Mechanistic studies are starting to shed light on the collaboration between mammalian NBR1 and p62 in the autophagic degradation of protein aggregates (aggrephagy). Several domains of NBR1 are involved in cargo recognition, and the list of known substrates for NBR1-mediated selective autophagy is increasing. Lastly, roles of NBR1 in human diseases such as proteinopathies and cancer are emerging.

Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0–4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

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Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by TBK1 (Tank-binding kinase 1) activating a LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting expression of pro-inflammatory genes and proteins. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

Introduction: NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), a member of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases, has been shown to be an important regulator of multiple proteins involved in cancer development, including the tumor suppressor PTEN and the proto-oncogene MDM2. Furthermore, NEDD4 has been shown to be overexpressed and act as an oncogene in multiple cancer types. We have previously shown that NEDD4 is overexpressed in colorectal cancer (CRC) and that it can promote growth of colon cancer cells independently of PTEN and PI3K/AKT signaling. In the present study, we investigated the role of NEDD4 in regulating the PTEN/PI3K pathway and the MDM2/p53 axis in CRC. Materials and Methods: The CRISPR/Cas9 system was applied to generate a Caco2 NEDD4 knockout cell line, and NEDD4 knockdown was performed in LS174T and SW480 cells. Gene expression profiles of 412 primary CRCs, 51 normal mucosa samples, 38 CRC cell lines, as well as the Caco2 parental and NEDD4 knockout cell lines, were generated using exon-resolution Affymetrix Human Exon Arrays or Human Transcriptome Arrays. The NEDD4 expression level was correlated with the mutation status of KRAS, PTEN, TP53, PIK3CA, BRAF and NRAS. Western blotting was used to detect and quantify NEDD4, PTEN and MDM2 protein levels. Results and Discussions: Gene expression analysis of the patient material confirmed our previous study that NEDD4 is significantly upregulated in CRC as compared to normal colonic mucosa. There was no correlation between NEDD4 expression and mutations in KRAS, BRAF, NRAS, PTEN, PIK3CA and TP53. CRISPR/Cas9-mediated NEDD4 knockout in Caco2 cells resulted in a reduction (P <0.01) in both the PTEN and MDM2 protein levels as compared to control cells. Gene set analysis showed that PI3K/AKT/MTOR signaling was upregulated in the NEDD4 knockout cells as compared to control cells. siRNA-mediated depletion of NEDD4 in LS174T cells was associated with reduced levels of MDM2, but did not affect the PTEN protein level. In SW480 cells, depletion of NEDD4 affected neither the MDM2 nor the PTEN protein level. By analyzing the expression of NEDD4, PTEN and MDM2 in 38 CRC cell lines by Western blotting, a positive correlation (P<0.01) was observed between NEDD4 and PTEN protein, while there was no significant correlation between NEDD4 and MDM2. Conclusion: The data show that NEDD4 is significantly upregulated in CRC, and that NEDD4 expression correlates with PTEN expression. The data further suggest that NEDD4 has the ability to regulate the PTEN and MDM2 protein levels in colon cancer cells in a cell line-specific manner. Citation Format: Lars M. Knudsen, Anita Sveen, Christer A. Andreassen, Christian H. Bergsland, Ina A. Eilertsen, Nikoline L. Rasmussen, Max Z. Totland, Peter W. Eide, Jarle Bruun, Ragnhild A. Lothe, Edward Leithe. Role of the E3 ubiquitin ligase NEDD4 in the regulation of PTEN and MDM2 in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1433.

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