Curr. Issues Mol. Biol. (2020) 35: 109-126. Roles of Ubiquitination and SUMOylation in the Regulation of Angiogenesis Andrea Rabellino1*, Cristina Andreani2 and Pier Paolo Scaglioni2 1QIMR Berghofer Medical Research Institute, Brisbane City, Queensland, Australia. 2Department of Internal Medicine, Hematology and Oncology; University of Cincinnati, Cincinnati, OH, USA. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.035.109 Abstract is tumorigenesis-induced angiogenesis, during Te generation of new blood vessels from the which hypoxic and starved cancer cells activate existing vasculature is a dynamic and complex the molecular pathways involved in the formation mechanism known as angiogenesis. Angiogenesis of novel blood vessels, in order to supply nutri- occurs during the entire lifespan of vertebrates and ents and oxygen required for the tumour growth. participates in many physiological processes. Fur- Additionally, more than 70 diferent disorders have thermore, angiogenesis is also actively involved been associated to de novo angiogenesis including in many human diseases and disorders, including obesity, bacterial infections and AIDS (Carmeliet, cancer, obesity and infections. Several inter-con- 2003). nected molecular pathways regulate angiogenesis, At the molecular level, angiogenesis relays on and post-translational modifcations, such as phos- several pathways that cooperate in order to regulate phorylation, ubiquitination and SUMOylation, in a precise spatial and temporal order the process. tightly regulate these mechanisms and play a key In this context, post-translational modifcations role in the control of the process. Here, we describe (PTMs) play a central role in the regulation of these in detail the roles of ubiquitination and SUMOyla- events, infuencing the activation and stability of tion in the regulation of angiogenesis. many growth factors, membrane receptors and downstream signalling efector molecules. Here, we will focus on the role of ubiquitination and Introduction SUMOylation in the regulation of angiogenesis. Te growth of new blood vessels from the exist- ing vasculature is a process known as angiogenesis (Carmeliet, 2003; Ucuzian et al., 2010). In ver- Molecular basics of tebrates, angiogenesis occurs across the entire angiogenesis lifespan and participates in multiple physiological Blood vessels arise from endothelial precursor processes, such as pregnancy, embryonic develop- cells, in a process known as vasculogenesis. Further ment and wound healing. Moreover, many diseases stabilization of the new blood vessels, including can promote de novo angiogenesis, a process also their expansive growth and the formation of known as pathological angiogenesis or neo- collateral bridges is known as angiogenesis (Car- angiogenesis. In this regard, a well-known example meliet, 2003). During angiogenesis, a dynamic caister.com/cimb 109 Curr. Issues Mol. Biol. Vol. 35 314 | Rabellino et al. and complex crosstalk occurs between endothe- can regulate cell migration in response to VEGF. lial cells and the extracellular matrix in a tightly On the other hand, anti-angiogenic factors such as regulated manner in order to promote endothelial the Endostatins are potent inhibitors of endothelial cells proliferation and diferentiation, cytoskeletal cells migration counteracting the formation of focal reorganization and cell migration, and the forma- adhesions (Dixelius et al., 2003; Eriksson et al., tion of novel vessels (Carmeliet, 2003; Huang and 2003). Bao, 2004; Muñoz-Chápuli et al., 2004; Ucuzian et Typically, during endothelial cell migration, al., 2010). Endothelial cells, fbroblasts, platelets, cell proliferation is enhanced, while apoptosis is infammatory cells and cancer cells (Ucuzian et repressed. Generally, the apoptotic signals that al., 2010) can all act as sources of angiogenic fac- regulate angiogenesis and the fate of endothelial tors. Key pro-angiogenic factors are the Vascular cells are mediated by TNF-α and TGF-β signalling Endothelial Growth Factors (VEGF1–5) and their (Polunovsky et al., 1994; Choi and Ballermann, receptors (VEGFR1, VEGFR2 and VEGFR3), the 1995). During angiogenesis, however, apoptotic Placental Growth Factors (PlGFs), the Fibroblast pathways are inhibited by the crosstalk between Growth Factors (FGF1 and FGF2) and FGF Integrins, VEGF and FGF cascades that converge receptors (FGFR1–4), the Transforming Growth toward the activation of the AKT pathway (Gerber Factor (TGF-β) family, the Tumour Necrosis et al., 1998). Other signalling pathways involved in Factor (TNF-α), the family of the Angiopoietins angiogenesis include WNT signalling (Dufourcq et (ANG1 and ANG2) and the TIE-1 and -2 recep- al., 2002), and the pathways activated by cytokines, tors, Ephrins and Leptins (Carmeliet, 2003; Huang such as Pleiotrophin and Midkine (Stoica et al., and Bao, 2004; Ucuzian et al., 2010). On the 2001, 2002), and oestrogens (Hyder et al., 1996). other hand, anti-angiogenic factors include the Because hypoxia is an important factor in angio- Trombospondins (TSP1–4 and TSP-5/COMP), genesis, also Hypoxia-Inducible Factors (HIFs) Angiostatins and Endostatins (Huang and Bao, play a fundamental role in neo-angiogenesis during 2004). Moreover, other players may diferentially tumour development (Pugh and Ratclife, 2003). contribute to the control of angiogenesis, like the HIF is a basic helix–loop–helix heterodimeric Matrix Metalloproteinases (MMPs), Integrins, and transcription factor that under hypoxic condition the extracellular matrix (ECM) (Kessenbrock et al., binds to hypoxic response elements (HREs) of 2010). Tese factors activate several downstream the DNA inducing the transcription of a series of signalling pathways. For example, VEGF, and hypoxia-related genes, many of which are involved similarly FGF, mainly activate the ERK/MAPK in angiogenesis (Semenza, 2000; Wenger, 2002). pathway (Larsson et al., 1999; Cross et al., 2000; Wu For example, VEGF transcription is directly up- et al., 2000), leading to the transcription of master regulated by HIF activity in hypoxic conditions genes involved in cell proliferation, such as MYC, (Levy et al., 1998; Pugh and Ratclife, 2003; Zhang ELK-1, FOS, etc. (Muñoz-Chápuli et al., 2004). On et al., 2012; De Francesco et al., 2013). Accordingly, the other end, VEGF can also act independently of Hif-1α knock out mice show abnormal vascular the ERK/MAPK cascade by activating other path- development and embryonic lethality (Maltepe et ways such as the STAT signalling (Muñoz-Chápuli al., 1997; Kotch et al., 1999). et al., 2004). Interestingly, additional stimuli can cooperate with angiogenic factors. Accordingly, Nitric Oxide (NO) is able to potentiate the VEGF- PTMs in angiogenesis dependent activation of the angiogenic pathways PTMs are a series of covalent modifcations that (Donnini and Ziche, 2002). VEGF also directly occur following protein synthesis, and regulate controls the migration of endothelial cells during protein activity, turnover and/or localization. Te angiogenesis activating the RHO GTPases RHO most common PTMs include phosphorylation, and RC, which are required for cell motility acetylation, glycosylation, ubiquitination and and the formation of focal adhesions (Soga et al., SUMOylation. Every PTM is strictly regulated 2001a,b). Moreover, other factors, such as the by several molecular mechanisms and feedback Protein Kinase C (PKC) (Yamamura et al., 1996), loops. Interestingly, every single PTM described or the receptor NOTCH (Hellström et al., 2007), so far participates in the regulation and control caister.com/cimb 110 Curr. Issues Mol. Biol. Vol. 35 Ubiquitin and SUMO in Angiogenesis | 315 of angiogenesis (Rahimi and Costello, 2015). In so far, including RanBP2, PC2, TOPORS and the this review, we will focus on ubiquitination and PIAS family (Rabellino et al., 2017). SUMOylation, and will describe how these PTMs Both ubiquitination and SUMOylation start work and impact angiogenesis. with the atachment of a single Ub or SUMO to the target protein: these mono-ubiquitination/ SUMOylation events have several repercussions Ubiquitination and SUMOylation on the fate of the target. Moreover, both Ub and in angiogenesis SUMO ofen form complex branched chains, and Ubiquitination and SUMOylation are PTMs the complexity and/or the length of the chains that regulate the activity and fate of a plethora will determine the fate of the modifed-target. For of proteins (Clague et al., 2015; Hendriks et al., example, Ub contains seven K residues that can be 2015). Both ubiquitination and SUMOylation ubiquitinated, thus participating to the formation consist of the covalent binding of a small protein of complex and branched Ub chains (Kim et al., modifer (ubiquitin, Ub hereafer, or Small 2011; Wagner et al., 2011). Ubiquitin-like Modifer, SUMO hereafer) to one Owing to the presence of multiple SUMO paral- or multiple lysine (K) residues of a target protein. ogs, the SUMOylation machinery is more complex Both processes require three consecutive steps than ubiquitination. In vertebrates, fve diferent (Fig. 18.1), sequentially catalysed by E1, E2 and SUMO genes exist and they encode for 5 diferent E3 ligases (Swatek and Komander, 2016; Rabellino SUMO proteins (SUMO1–5): SUMO1, SUMO2 et al., 2017). While for ubiquitination, a variety of and SUMO3 are ubiquitously expressed, while E1–3 ligases are known, for SUMOylation, only one SUMO4 and SUMO5 are tissue specifc and not E1 (SAE1/2) and one E2 (UBC9) are known, and well characterized