The role of ubiquitination and deubiquitination in tumor invasion and metastasis

Ubiquitination is vital for multiple cellular processes via dynamic modulation of proteins related to cell growth, proliferation, and survival. Of the ubiquitination system components, E3 ubiquitin ligases and deubiquitinases have the most prominent roles in modulating tumor metastasis. This review will briefly summarize the observations and underlying mechanisms of multiple E3 ubiquitin ligases and deubiquitinases to regulate tumor metastasis. Further, we will discuss the relationship and importance between ubiquitination components and tumor progression.     

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Abstract

Gα_q-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gα_q-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions: Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.     

The role of atypical ubiquitination in cell regulation

Ubiquitination is a type of intracellular proteins post-translational modification (PTM) characterized by covalent attachment of ubiquitin molecules to target proteins. This includes monoubiquitination (attachment of one ubiquitin molecule), multiple monoubiquitination also known as multiubiquitination (attachment of several monomeric ubiquitin molecules to a target protein), and polyubiquitination (attachment of ubiquitin chains consisting of several, most frequently four ubiquitin monomers to a target protein). In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-linked polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins (not necessarily) targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes including immune response, genome stability, signal transduction, etc. Alterations of ubiquitination machinery is crucial for development of serious diseases.     

Control of DNA polymerase lambda stability by phosphorylation and ubiquitination during the cell cycle

DNA polymerase (Pol) lambda is a DNA repair enzyme involved in base excision repair, non-homologous end joining and translesion synthesis. Recently, we identified Pol lambda as an interaction partner of cyclin-dependent kinase 2 (CDK2) that is central to the cell cycle G1/S transition and S-phase progression. This interaction leads to in vitro phosphorylation of Pol lambda, and its in vivo phosphorylation pattern during cell cycle progression mimics the modulation of CDK2/cyclin A. Here, we identify several phosphorylation sites of Pol lambda. Experiments with phosphorylation-defective mutants suggest that phosphorylation of Thr 553 is important for maintaining Pol lambda stability, as it is targeted to the proteasomal degradation pathway through ubiquitination unless this residue is phosphorylated. In particular, Pol lambda is stabilized during cell cycle progression in the late S and G2 phases. This most likely allows Pol lambda to correctly conduct repair of damaged DNA during and after S phase.     

Spatial regulation of exocytosis and cell polarity: yeast as a model for animal cells

Exocytosis is the major mechanism by which new membrane components are delivered to the cell surface. In most, if not all, eukaryotic cells this is also a highly spatially regulated process that is tightly coordinated with the overall polarity of a cell. The Rho/Cdc42 family of GTPases and the lethal giant larvae/Sro7 family are two highly conserved families of proteins which appear to have dual functions both in cell polarity and exocytosis. Analysis of their functions has begun to unravel the coordination between these processes and propose a model for polarized vesicle docking and fusion at the site of asymmetric cell growth.     

The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells

Ubiquitin-specific protease 11 (USP11) has been implicated in the regulation of DNA repair, apoptosis, signal transduction and cell cycle. It belongs to a USP subfamily of deubiquitinases. Although previous research has shown that USP11 overexpression is frequently found in melanoma and is correlated with a poor prognosis, the potential molecular mechanism of USP11 in melanoma remains indefinitive. Here, we report that USP11 and NONO colocalize and interact with each other in the nucleus of melanoma cells. As a result, the knockdown of USP11 decreases NONO levels. Whereas, overexpression of USP11 increases NONO levels in a dose-dependent manner. Furthermore, we reveal that USP11 protects NONO protein from proteasome-mediated degradation by removing poly-ubiquitin chains conjugated onto NONO. Functionally, USP11 mediated melanoma cell proliferation via the regulation of NONO levels because ablation of USP11 inhibits the proliferation which could be rescued by ectopic expression of NONO protein. Moreover, a significant positive correlation between USP11 and NONO concentrations was found in clinical melanoma samples. Collectively, these results demonstrate that USP11 is a new deubiquitinase of NONO and that the signalling axis of USP11-NONO is significantly involved in melanoma proliferation.     

The emerging role of USP29 in cancer and other diseases

Reversible protein ubiquitination is a key process for maintaining cellular homeostasis. Deubiquitinases, which can cleave ubiquitin from substrate proteins, have been reported to be deeply involved in disease progression ranging from oncology to neurological diseases. The human genome encodes approximately 100 deubiquitinases, most of which are poorly characterized. One of the well-characterized deubiquitases is ubiquitin-specific protease 29 (USP29), which is often upregulated in pathological tissues and plays important roles in the progression of different diseases. Moreover, several studies have shown that deletion of Usp29 in mice does not cause visible growth and developmental defects, indicating that USP29 may be an ideal therapeutic target. In this review, we provide a comprehensive summary of the important roles and regulatory mechanisms of USP29 in cancer and other diseases, which may help us better understand its biological functions and improve future studies to construct suitable USP29-targeted therapy systems.     

The role of gap junctions in trophoblastic cell fusion in the guinea-pig placenta

Summary Fusion of cytotrophoblast cells in the guinea-pig placenta occurs at regions of plasma membrane interdigitation where the cells are attached to one another by complex arrays of gap junctions and desmosomes. Fusion begins at the gap junctions, which are lost in this process. The desmosomes play no obvious part in the fusion mechanism and remain after fusion as sites of attachment of syncytiotrophoblast membrane to itself. It is proposed that a major role of gap junctions in placental development is to bring trophoblast plasma membranes into a close relationship which may act as a starting point for cell fusion.     

Usp7 and Uhrf1 control ubiquitination and stability of the maintenance DNA methyltransferase Dnmt1

In mammals Dnmt1 is the DNA methyltransferase chiefly responsible for maintaining genomic methylation patterns through DNA replication cycles, but how its maintenance activity is controlled is still not well understood. Interestingly, Uhrf1, a crucial cofactor for maintenance of DNA methylation by Dnmt1, is endowed with E3 ubiquitin ligase activity. Here, we show that both Dnmt1 and Uhrf1 coprecipitate with ubiquitin specific peptidase 7 (Usp7), a de-ubiquitinating enzyme. Overexpression of Uhrf1 and Usp7 resulted in opposite changes in the ubiquitination status and stability of Dnmt1. Our findings suggest that, by balancing Dnmt1 ubiquitination, Usp7 and Uhrf1 fine tune Dnmt1 stability.     

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.     

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.     

The role of EGF receptor ubiquitination in regulating its intracellular traffic

Progression of activated EGF receptor (EGFR) through the endocytic pathway regulates EGFR signaling. Here we show that a non-ubiquitinated EGFR mutant, unable to bind the endosomal-sorting complex required for transport (ESCRT) component, Hrs, is not efficiently targeted onto intraluminal vesicles (ILVs) of multivesicular endosomes/bodies (MVBs). Moreover, ubiquitination and ESCRT engagement of activated EGFR are required for EGF-stimulated ILV formation. Non-ubiquitinated EGFRs enter clathrin-coated tubules emanating from MVBs and show enhanced recycling to the plasma membrane, compared to wild-type EGFR.     

Caveolin-1 in cell polarization and directional migration

Migration is a complex process in which cells move in a given direction either in response to changes in the extracellular environment or as a consequence of an intrinsic propensity for directional movement. Migration plays key roles in many physiological and pathological processes, including development, angiogenesis, tissue regeneration and metastasis. An important role in migration is played by caveolin-1 and caveolae. Caveolae compartmentalize intracellular signalling pathways to orchestrate cell migration. Caveolin-1 presents a polarized distribution in migrating cells and is linked to the cytoskeleton, and changes in its expression modulate migration. Although there are some discrepancies regarding the regulatory effect of caveolin-1, most studies show that it promotes cell movement and polarity. The importance of caveolin-1 has recently been reinforced by studies with Cav1(-/-) cells, which indicate that it establishes polarity during directional migration by coordinating Src kinase and Rho GTPase signalling.     

Deciphering the functional role of endothelial junctions by using in vivo models

Endothelial cell-to-cell junctions are vital for the formation and integrity of blood vessels. The main adhesive junctional complexes in endothelial cells, adherens junctions and tight junctions, are formed by transmembrane adhesive proteins that are linked to intracellular signalling partners and cytoskeletal-binding proteins. Gene inactivation and blocking antibodies in mouse models have revealed some of the functions of the individual junctional components in vivo, and are increasing our understanding of the functional role of endothelial cell junctions in angiogenesis and vascular homeostasis. Adherens-junction organization is required for correct vascular morphogenesis during embryo development. By contrast, the data available suggest that tight-junction proteins are not essential for vascular development but are necessary for endothelial barrier function.     

The role of cytoskeletal components in the maintenance of intercellular junctions in an insect

Summary Accessory glands of the cockroach are composed of secretory and supportive cells, the latter providing a skeleton-like framework of attentuated cytoplasmic processes into which the former are positioned. These two cell types are associated with one another laterally by adhaering, pleated septate, and gap junctions. Hemi-adhaerens junctions are also found on both luminal and basal surfaces of the gland; the former are associated with the cuticular lining of the lumen and the latter with extracellular matrix. The adhering and septate junctions are flanked by both filaments and microtubules; the former insert into the junctional membranes and are actin-like, binding both rhodamine-conjugated phalloidin and the S₁ subfragment of rabbit heavy meromyosin. The role of this cytoskeletal protein with the cellular junctions has been explored by treatment with a disruptive agent, cytochalasin D. Dissociation of actin leads to changes in septate junctions and in microtubular distribution. This suggests that the latter act as anchors for the actin filaments which, in turn, appear bound to certain of the intramembranous junctional components.Supported by a Conicet/Royal Society Visiting Fellowship     

The importance of regulatory ubiquitination in cancer and metastasis

Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis.