Neddylation - The copyright of this thesis vests in the author. No quotation from it

Chapter 1: Introduction

1.5 Neddylation

is similar to ubiquitination and the pathways crosstalk in their activities within the cell. We will review neddylation and current knowledge on it as an emerging pathway in terms of PTMs but also as an emerging target for cancer treatment.

Model organism experiments have demonstrated that neddylation is an essential pathway because dysregulation or knockout of NEDD8 pathway components leads to lethality in some organisms and developmental defects in others (Tateishi et al., 2001; Kurz et al., 2002; Figueroa et al., 2005; Dorfman et al., 2009; Hosp et al., 2014). Additionally, neddylation has been implicated in transcription, DNA replication, cell growth, cell proliferation, DNA damage repair and apoptosis (Chairatvit and Ngamkitidechakul, 2007; Jones et al., 2008; Soucy et al., 2009; Merbl et al., 2013). As such, it has been implicated in several diseases including neurodegenerative, cardiovascular diseases and cancer (Watson, Irwin and Ohh, 2011; Chen, Neve and Liu, 2012; Duncan et al., 2012; Kandala, Kim and Su, 2014). Neddylation also plays a role in ubiquitin CRL activity, with cullin protein neddylation important for the conformational changes that enhance ubiquitin substrate modification and related activities (Saha A, 2008). The cullin family of proteins: cullin 1, 2, 3, 4A, 4B, 5, 7, except for p53-associated parkin- like cytoplasmic protein (PARC), are among the proteins targeted for neddylation (Skaar et al., 2007;

Kim et al., 2008; Sarikas, Hartmann and Pan, 2011).

As mentioned previously, cullin proteins are scaffolding molecules of the ubiquitin CRLs which target a variety of proteins for ubiquitination leading to proteasomal degradation, among other activities (Petroski and Deshaies, 2005). The targets and components of CRLs include regulators of the cell cycle, receptors, tumour suppressors, oncoproteins and knockdown/knockout of cullin proteins can lead to Figure 15. Schematic diagram of the neddylation pathway and DCUN1D1 cullin neddylation.

Showing the neddylation pathway and an example of the E3 ligase DCUN1D1 and its known substrates, cullin 1, 2, 3, 4A, 4B and cullin 5. The cullin proteins are scaffolding molecules of the cullin RING E3 ligases (CRLs). It also includes inhibitors of neddylation, namely, CAND1 (left) which binds inactive cullin proteins prior to neddylation and DEN1 or UCH L3 (right) which are known to process precursor NEDD8 and remove NEDD8 from target proteins.

dysregulation of these proteins or their functions (Sarikas, Hartmann and Pan, 2011). Significantly, cullin 3 knockout in C. elegans results in defective embryogenesis and knockout of our protein of interest DCUN1D1, results in developmental abnormalities in mice (Kurz et al., 2002; G. Huang et al., 2011). Additionally, the following proteins have been described as neddylation substrates including p53, MDM2, pVHL, several ribosomal proteins, EGFR and some ubiquitin E3 ligases (Kamura et al., 1999; Stickle et al., 2004; Xirodimas et al., 2004, 2008; Oved et al., 2006; Jones et al., 2008; Watson, 2010). Table 7 below provides a summary of the neddylation substrates that have been identified, including details on p53 and MDM2, which are more well understood. P53 and MDM2 are a NEDD8- substrate and E3 ligase pair, while p53 is also regulated by other NEDD8 substrates such as ribosomal proteins (Xirodimas et al., 2004; Sundqvist et al., 2009; Watson, 2010). This provides insights into the range of mechanisms of action of neddylation and the functions it may perform, including the regulation of protein-protein interactions and regulating protein stability (Figure 16). This is evident also in its role in cancer.

Table 7: Showing neddylation substrates and neddylation-related functions

Neddylation substrate Function Neddylation substrate

Function

P53 MDM2: Mediates p53

neddylation Tip60: Inhibits p53- MDM2-mediated neddylation

NUB1: Decreases p53 neddylation leading to nuclear export

FBXW011: Promotes p53 neddylation inhibiting transactivation activity

HIF1α Neddylation results in HIF1α stability

MDM2 MDM2: undergoes

autoneddylation P53: MDM2 neddylation substrate

TAp73β: MDM2 neddylation substrate L11: MDM2 neddylation substrate

RCAN1 Neddylation results in RCAN1 stability

RPL11 Neddylation results in

RPL11 stability and localization

TGF β Receptor II

c-Cbl mediates TβRII neddylation and stabilization RPL5, RPL6, RPL7, RPL7a,

RPL8, RPL9, RPL10a, RPL11, RPL12, RPL13, RPL14, RPL17, RPL18, RPL21, RPL23, RPL24, RPL26, RPL27, RPL29, RPL30, RPL31, RPL35a, RPS2, RPS3, RPS4, RPS6, RPS7, RPS8, RPS11, RPS13, RPS14, RPS15a, RPS16, RPS20, RPS23, RPS26

Targets of neddylation determined from proteomics

CK1α First description of kinase neddylation

PARKIN Undergoes

autoneddylation PINK1 is its E3 ligase

SHC Undergoes neddylation

impacting T cell activity

PINK1 Neddylation results in

PINK1 stability

HUR MDM2 mediates HUR

neddylation affecting its stability

BRAP2 Brap2 neddylation

impacts NF-κB nuclear translocation

Histone H4 RNF111 mediates histone H4 neddylation regulating DNA damage

XIAP Undergoes

autoneddylation

E2F1 Neddylation controls transcriptional target specificity and induction of apoptosis

AICD Neddylation results in the

inhibition of its transcriptional activity

ML3 Undergoes neddylation

Neddylation is dysregulated in multiple cancers including liver, lung, oral squamous cell carcinoma and PCa (Soucy et al., 2009; Lin et al., 2010; Milhollen et al., 2010; Swords et al., 2010; Wei et al., 2012;

Shah et al., 2016). Additionally, some of the identified neddylation substrates have been established to play a role in cancer. The tumour suppressors p53 and VHL are dysregulated in many cancers through mutations, PTMs, dysregulation in expression levels and they have been shown to undergo neddylation (Stickle et al., 2004; Xirodimas et al., 2004; Meek and Anderson, 2009; Bieging, Mello and Attardi, 2014; Gossage, Eisen and Maher, 2015). P53 neddylation is regulated by its E3 ligase MDM2, by RPL11, Tip60, NUB1 and FBXW011 (Table 7) affecting its stability, nuclear translocation and transcriptional activity (Xirodimas et al., 2004; Abida et al., 2007; Dohmesen, Koeppel and Dobbelstein, 2008; Sundqvist et al., 2009; Liu and Xirodimas, 2010). However, no direct association has been made between p53 neddylation and cancer thus far. VHL neddylation on the other hand inhibits cullin 2 binding and the role of VHL neddylation in the disruption of fibronectin extracellular matrix assembly is associated with inhibiting tumour development (Stickle et al., 2004; Heir et al., 2013). Several other NEDD8 substrates have been associated with cancer growth and development, independent of neddylation, but affecting different “Hallmarks of Cancer” such as MDM2, HIF1α and BCA3 (Kitching et al., 2003; Vaupel, 2004; Leung and Ngan, 2010; Karni-Schmidt, Lokshin and Prives, 2016; D. Ma et al., 2018). It is however, the development and success of the NAE inhibitor (MLN4924) that highlights the importance of the neddylation pathway in cancer.

The NAE inhibitor MLN4924 has been shown to have potent anti-cancer activity, particularly in myelomas and it is undergoing phase II clinical trials in other cancers (Milhollen et al., 2010; Swords et al., 2010, 2015; Luo et al., 2012; Wei et al., 2012). However, several side effects have been associated with its use in cancer treatment (Milhollen et al., 2010; Swords et al., 2010, 2015; Luo et al., 2012; Wei et al., 2012). In fact, p53 has been tested for the protection of normal cells from MLN4924 treatment side effects, relying on the role of p53 in cell stress regulation (Malhab et al., 2016). MLN4924 has been shown to activate p53 in cancer cells expressing wildtype p53 through deneddylation of some of its regulators, however, cells not expressing p53 or expressing a mutated form of it were still sensitive to MLN4924 (Malhab et al., 2016). Therefore, it has been proposed that, regulation of p53 neddylation may be useful in MLN4924 treatment sensitization when used in combination with p53 inhibitors or it could be used to regulate MLN4924 toxicity in managing side effects (Malhab et al., 2016). This supports our hypothesis that targeting the neddylation pathway may provide cancer treatment efficiencies, with potentially reduced side effects. Studies performed previously in our lab demonstrated the role of DCUN1D1 in PCa using in vitro and in vivo analyses (Vava and Zerbini, 2014). We also tested drugs to identify DCUN1D1-specific potential inhibitors of PCa and we propose that targeting neddylation, using the downstream DCUN1D1 E3 ligase, may retain potency in cancer treatment and perhaps result in reduced side effects. We therefore want to understand the mechanisms involved in DCUN1D1’s activity in PCa inhibition, using proteomics.

Proteomics has already been used to identify some neddylation substrates through affinity purification-based MS analysis and SILAC quantitative proteomics. The studies employed different approaches where affinity purification-based MS was performed following pulldown of proteins bound to NEDD8, after the overexpression of GST-NEDD8 in HEK293 cell lines (Jones et al., 2008). The LTQ mass spectrometer was used and 496 proteins were identified, that were involved in cell cycle regulation, chromatin structure and organization, transcription, DNA replication and DNA damage repair (Jones et al., 2008). The study by Liao et al., 2011 used SILAC-based quantitative proteomics analysis of proteins that were stabilized following MLN4924 treatment of A375 melanoma cells. The

objective was to identify new downstream targets of CRL substrates by inhibiting neddylation, preventing cullin protein neddylation and ubiquitin CRL assembly (Liao et al., 2011). This would lead to the accumulation of proteins normally degraded by the 26S proteasome (Liao et al., 2011). Using the LTQ Orbitrap Velos, 5122 - 6012 proteins were identified over the different time points that they analysed and CRL substrates involved in the cell cycle, DNA damage repair and the ubiquitin pathway were identified (Liao et al., 2011). Additionally, using the overexpression of tandem affinity purification (TAP)-tagged NEDD8 in Hela cells and the Q-Star Pulsar XL mass spectrometer, several ribosomal proteins were identified as targets for neddylation (Table 7) (Xirodimas et al., 2008).

Therefore, neddylation is rapidly emerging as a critical PTM, with many substrates being identified that lead to increasingly complicated cellular activities. Apart from understanding the role that NEDD8 modification plays on protein substrates, one of the biggest questions surrounding neddylation is whether the substrates identified thus far are true/putative neddylation substrates or are artefacts of the overexpression of NEDD8, which leads to NEDD8 transfer being mediated by ubiquitin pathway components. NEDD8 and ubiquitin are 60% identical in terms of amino acid sequence and Leidecker et al., 2012 demonstrated that the UAE UBE1 was able to mediate neddylation, especially of non-cullin molecules under conditions of stress (Kumar, Yoshida and Noda, 1993; Leidecker et al., 2012).

However, it is not uncommon for proteins/enzymes to have overlapping activity as demonstrated above with the other UBLs. In fact, some of the neddylation substrates that have been identified are characterized ubiquitin E3 ligases (MDM2 and PARKIN) that have been shown to mediate neddylation of substrates or to undergo autoneddylation (Fang, 2000; Xirodimas et al., 2004; Watson et al., 2006;

Choo et al., 2012; Seirafi, Kozlov and Gehring, 2015).

Conversely, it has been proposed that the above-mentioned, maybe a bona fide cellular regulatory mechanism demonstrating NEDD8 and ubiquitin crosstalk. Evidence for this can be obtained from how in cases like EGFR, monoubiquitination preceded NEDD8 modification by the same E3 ligase, c-Cbl, suggesting that monoubiquitination may be triggering neddylation (Oved et al., 2006). Additionally, Xirodimas et al., 2008 identified ubiquitin as a NEDD8 substrate using MS, while Leidecke et al., 2012 described mixed chains of ubiquitin and NEDD8 under conditions of stress (Xirodimas et al., 2008;

Leidecker et al., 2012). Therefore, neddylation is displaying characteristic molecular level functions and mechanisms that mimic that of ubiquitin, suggesting that it may be as important to cellular function as ubiquitination. Although the neddylation pathway, its substrates and the methods of identification raise interesting questions, there are a variety of pathways that have been implicated in neddylation and neddylation has been demonstrated to be essential, as demonstrated by in vivo studies (Kurz et al., 2002; G. Huang et al., 2011).

We have studied one of the neddylation E3 ligases, DCUN1D1, and identified it to play a key role in PCa in vitro and in vivo (Vava and Zerbini, 2014). We will use proteomics to understand its mechanism of action including attempting to identify any non-cullin proteins that it may be targeting but we begin with a review of what is currently understood about DCUN1D1.

Dalam dokumen The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. (Halaman 49-55)